Anti-ceacam6 antibodies and uses thereof

ABSTRACT

The present invention provides recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions that are specific for human and  Macaca fascicularis  CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6, CD66c, Non-specific crossreacting antigen, NCA, NCA-50/90), and which do not significantly cross-react with the closely related human CEACAM1, human CEACAM3, and human CEACAM5. The invention further provides methods to generate this kind of antibodies. The antibodies, accordingly, can be used to treat cancer and other disorders and conditions associated with expression of the CEACAM6. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.

The present invention provides recombinant antigen-binding regions andantibodies and functional fragments containing such antigen-bindingregions that are specific for human and Macaca fascicularis CEACAM6(Carcinoembryonic antigen-related cell adhesion molecule 6, CD66c,Non-specific crossreacting antigen, NCA, NCA-50/90), and which thereforedo not significantly cross-react with the closely related human CEACAM1,human CEACAM3, and human CEACAM5. The invention further provides methodsto generate this kind of antibodies.

The antibodies, accordingly, can be used to treat cancer and otherdisorders and conditions associated with the expression of the CEACAM6.The invention also provides nucleic acid sequences encoding theforegoing antibodies, vectors containing the same, pharmaceuticalcompositions and kits with instructions for use.

BACKGROUND OF THE INVENTION

Antibody-based therapy is an effective and clinically establishedtreatment of various cancers, including solid tumors. For example,HERCEPTIN® has been used successfully to treat breast cancer andRITUXAN® is effective in B-cell related cancer types. Central to thedevelopment of a novel successful antibody-based therapy is theisolation of antibodies against cell-surface proteins found to bepreferentially expressed on target cells (e.g. cancer cells, immunecells etc) that are able to functionally modify the activity of thecorresponding receptor.

Antibody blockade of immune checkpoint molecules for immune cellactivation and thus for immunotherapy of cancer is a clinicallyvalidated approach. In 2011 the CTLA-4 blocking antibody Ipilimumab hasbeen approved by the FDA for the 2nd line therapy of metastatic melanoma(Yervoy). Another example is the blockade of the PD-1/PD-L1 axis forwhich several drugs are either approved or currently under clinicaldevelopment and for which impressive clinical responses have beenreported in melanoma, RCC and lung cancer (Henick et al., Expert OpinTher Targets. 2014 December; 18(12):1407-20)).

Proteins of the Carcinoembryonic antigen-related cell adhesion molecule(CEACAM) family belong to the immunoglobulin (Ig) supergene family andgenerally exhibit a variable (V)-like domain identified as the N domain.The N domain is followed by either none or up to six constant C2-like Igdomains (termed A or B). These extracellular domains are required forCEACAM functionality as homo- and heterophilic intercellular adhesionmolecules (Obrinck, Curr Opin Cell Biol. 1997 October; 9(5):616-26) oras human and rodent pathogen receptors (Kuespert et al., Curr Opin CellBiol. 2006 October; 18(5):565-71; Voges et al., PLoS One.2012;7(6):e39908). CEACAM receptors associate as dimers or oligomers andmultiple associations with other partners at the membrane andconsequently modulate important functions. In addition to theirexpression in human tissues, the CEACAM gene family is highly conservedin 27 other mammalian species and is best described in mouse, rat,cattle, dog, platypus and opossum (Kammerer and Zimmermann, BMC Biol.2010 Feb 4; 8:12). The best characterized biological function of CEACAMsis the support of cell-cell adhesion through their homo- andheterophilic interactions, including a role in the differentiation andformation of a three-dimensional tissue structure, angiogenesis,apoptosis, tumor suppression, and metastasis. (Kuespert et al., CurrOpin Cell Biol. 2006 October; 18(5):565-71). More details on the familymembers are described in other reviews (Horst and Wagener, Handb ExpPharmacol. 2004;(165): 283-341; Gray-Owen and Blumberg, Nat Rev Immunol.2006 June; 6(6):433-46).

CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6,CD66c, Non-specific crossreacting antigen, NCA, NCA-50/90) is aglycosylphosphatidylinositol (GPI)-linked cell surface protein with oneN-domain and 2 C2-like domains which mediate a number of possible cis ortrans directed interactions of CEACAM proteins through theirextracellular domains with a variety of membrane receptors, a few ofwhich have been identified. (Beauchemin and Arabzadeh, Cancer MetastasisRev. 2013 December; 32(3-4):643-71).

CEACAM6 is expressed in a variety of epithelia of normal human tissuesuch as colon (Blumenthal et al., BMC Cancer, 2007, Jan. 3; 7:2.), lung(Kolla et al., Am J Physiol Lung Cell Mol Physiol 296: L1019-L1030) andgranulocytes (Kuroki et al., Biochem Biophys Res Commun. 1992 Jan. 31;182(2):501-6). In the granulocytic lineage CEACAM6 was expressed at allstages of granulocytic maturation except for the early lineage-committedprecursor cell (Strickland et al., J Pathol. 2009 July; 218(3):380-90);Scholzel et al., American Journal of Pathology, 156 (2), 595-605).CEACAM6 is not expressed in rodents. (Beauchemin et al., Exp Cell Res.1999 Nov. 1; 252(2):243-9).

CEACAM6 expression has been described for several cancers. In coloncancer CEACAM6 is upregulated in 55% of the cases and an independentprognostic factor allowing subdivision of patients into low andhigh-risk groups (Jantscheff et al., J Clin Oncol. 2003 Oct. 1;21(19):3638-46). In pancreatic adenocarcinoma 92% (n=82) of analyzedspecimens were found to be positive while CEACAM6 expression was moreprevalent in high-grade than in low grade PanIN lesions (Duxbury et al.,Ann Surg. 2005 March; 241(3):491-6). This was confirmed in another studywhere >90% of invasive pancreatic adenocarcinomas (110 of 115 tested)showed a robust (over-) expression of CEACAM6 (Strickland et al., JPathol. 2009 July; 218(3):380-90). In addition, Blumenthal et al.reported CEACAM6 expression in breast tumors, in pancreatic tumors,ovarian adenocarcinomas, lung adenocarcinoma, lymph node metastases andmetastases from breast, colon and lung tumors. (Blumenthal et al., BMCCancer. 2007 Jan. 3; 7:2).

CEACAM6 expression in breast cancer was also reported by others (Maraqaet al., Clin Cancer Res. 2008 Jan. 15; 14(2):405-11; Poola et al., ClinCancer Res. 2006 Aug. 1;.12(15):4773-83; Balk-Moller et al., Am JPathol. 2014 April; 184(4):1198-208); Tsang et al., Breast Cancer ResTreat. 2013 November; 142(2):311-22). In addition CEACAM6 expression hasbeen reported in multiple myeloma (Witzens-Harig et al., Blood 2013 May30; 121(22):4493-503), gastric cancer (Deng et al., Genet Mol Res. 2014Sep. 26; 13(3):7686-97) and head and neck cancer (Cameron et al., MolCancer. 2012 Sep. 28; 11:74).

Experimental evidence supports a role for CEACAM6 as important regulatorof metastasis. Kim et al. have shown that attenuating CEACAM6 expressionin LoVo cells using a CEACAM6-specific siRNA or increasing itsexpression in HCT116 cells, respectively, impeded or augmented invasionthrough the extracellular matrix (Kim et al., Clin Chim Acta. 2013 Jan.16; 415:12-9). Suppression of CEACAM6 expression leads to elevatedE-cadherin promoter activity. Blumenthal et al. showed that CEACAM5 andCEACAM6 contributed to CRC metastatic dissemination which could beblocked by monoclonal antibodies in vivo. (Blumenthal et al., BMCCancer. 2007 Jan. 3; 7:2). Also it has been shown that CEACAM6 isexpressed in CD133-positive cells in colon cancer samples able to formstem cell-enriched colon spheres for which proliferation, clonogenicpotential, as well as in vivo tumorigenic potential were significantlyhampered upon its silencing (Gemei et al., Cancer. 2013 Feb. 15;119(4):729-38). In breast cancer it was shown that tamoxifen resistantsamples are CEACAM6 overexpressing and CEACAM6 was a significantpredictor of recurrence of the disease (Maraqa et al., Clin Cancer Res.2008 Jan. 15; 14(2):405-11). siRNA mediated CEACAM6 silencing in aMMU1-tamoxifen-resistant MCF7 cell derivative reversed endocrineresistance, anchorage independence of these cells and invasiveproperties (Lewis-Wambi et al., Eur J Cancer. 2008 August;44(12):1770-9). In lung adenocarcinoma CEACAM6 expression wassignificantly associated with adverse clinical outcome (Kobayashi etal., Br J Cancer. 2012 Nov. 6; 107(10):1745-53). In pancreatic cancerCEACAM6 silencing with siRNA reversed the acquired anoikis resistance ofMia(AR) pancreatic tumor cells. Overexpression of CEACAM6 in Capan2pancreatic cancer cells augmented gemcitabine resistance whereassiRNA-mediated suppression of CEACAM6 expression in BxPC3 cellschemosensitized them to the drug by modulating AKT activity in an Srcdependent manner (Duxbury et al., Cancer Res. 2004 Jun. 1;64(11):3987-93). These effects corresponded to increased invasiveness ofhigh CEACAM6 expressing cells exhibiting c-src activity and matrixmetalloproteinase (MMP9) expression (Duxbury et al., Br J Cancer. 2004Oct. 4; 91(7):1384-90).

T-cell responses against tumor-associated antigens have been describedin many tumors (Beckhove et al., J Clin Invest. 2004 July; 114(1):67-76;Choi et al., Blood. 2005 Mar. 1; 105(5):2132-4; Sommerfeldt et al.,Cancer Res. 2006 Aug. 15; 66(16):8258-65; Schmitz-Winnenthal et al.,Cancer Res. 2005 Nov. 1; 65(21):10079-87.; Jager et al., Proc Natl AcadSci USA. 2000 Apr. 25; 97(9):4760-5; Romero et al., Adv Immunol. 2006;92:187-224) and often cause an accumulation of tumor specific memory Tcells in lymphoid organs or in the blood (Choi et al., Blood. 2005 Mar.1; 105(5):2132-4; Feuerer et al., Nat Med. 2001 April; 7(4):452-8;Letsch et al., Cancer Res. 2003 Sep. 1; 63(17):5582-6). However, thecapacity of T cells to react against autologous tumor cells is generallylow (Horna and Sotomayor, Curr Cancer Drug Targets. 2007 February;7(1):41-53); Yang and Carbone, Adv Cancer Res. 2004; 92:13-27). Manytumors have the capacity to block effector functions of T cells whichcontributes to the limited activity of tumor immunotherapy. T-cellunresponsiveness against tumor cells has been demonstrated for a broadvariety of cancers (Pardoll, Nat Immunol. 2012 December;13(12):1129-32).

CEACAM6 also contributes to the regulation of CD8+T cell response.Recently, Witzens-Harig et al. demonstrated in multiple myelomaexpressing several CEACAM family members that treatment withanti-CEACAM6 mAbs or siRNA silencing CEACAM6 reinstated T cellreactivity against malignant plasma cells indicating a role for CEACAM6in CD8+T cell response regulation (Witzens-Harig et al., Blood 2013 May30; 121(22):4493-503). So far, a receptor for CEACAM6 on T cells has notbeen identified. However, co-culture of CEACAM6 positive myeloma cellswith T cells resulted in the modulation of T cell signaling eventsincluding an activation of SHP phosphatases by CEACAM6 ligation (Lin andWeiss, J Cell Sci. 2001 January; 114(Pt 2):243-4; Latour et al., MolCell Biol. 1997 August; 17(8):4434-41; Wen et al., J Immunol. 2010 Dec.1; 185(11):6413-9). CEACAM6 has no intrinsic signaling capacity, and itsinhibitory capacity is presumably mediated by binding to receptors onthe T cell surface. Such a receptor can be for example CEACAM1 for whichmechanism for the modulation of innate and adaptive immune responseshave been described. CEACAM1 (CD66a) possesses a cytoplasmic tailcontaining an immunoreceptor tyrosine-based inhibitory (ITIM) motif.CEACAM1 is stored in intracellular vesicles and upon T cell activationis rapidly (24 h to 72 h) externalized and expressed on the T cellsurfacewhere it mediates the blockade of T-cell effector functions afterhomo- or heterophilic binding to ligands expressed on target cells(Gray-Owen and Blumberg, Nat Rev Immunol. 2006 June; 6(6):433-46). Thenature of this binding is unknown and could be either homo- orheterophilic binding to other CEACAMs or binding to other components ofthe extracellular matrix, growth factor receptors, integrins, orcadherins. Homophilic interactions have been reported between CEACAM1and CEACAM1 (Ortenberg et al., Mol Cancer Ther. 2012 June;11(6):1300-10). Heterophilic CEACAM interactions have been described forexample between CEACAM1 and CEACAM5, and CEACAM6 and CEACAM8 (Cavallaroand Christofori, Nat Rev Cancer. 2004 February; 4(2):118-32).

As described above CEACAM6 is a very attractive target for therapeuticintervention in cancer immunotherapy. As noted, CEACAM6 is a member of afamily of highly homologous proteins. An antibody suitable for humantherapy, which is relieving immunosuppression of CEACAM6, must thereforebe able to distinguish between CEACAM6 and other paralogous proteinslike CEACAM1, CEACAM3, CEACAM5, which each display different functionsand tissue distributions, to restrict its mode of action andlocalization to CEACAM6 and to avoid unwanted adverse side effects.

As CEACAM6 is not only expressed on tumor cells but also on normaltissues (especially granulocytes but also epithelial cells of e.g. lungand gastrointestinal cells—Chan and Stanners, Mol Ther. 2004 June;9(6):775-85; Strickland et al., J Pathol. 2009 July; 218(3):380-90), itis absolutely crucial to be able to predict the adverse side effectprofile of the therapeutic antibody. This is all the more important,since the anticipated mode of action will be inhibition ofimmunosuppression, i.e. an immunoactivation, which can result in serioushazards (incident of CD28 superagonist TGN1412 trial; Suntharalingam etal., N Engl J Med. 2006 Sep. 7; 355(10):1018-28). So indirect effects onthe immune system on top of direct effects on granulocytes need to becarefully assessed. To enable the development of a human therapeuticantibody and a predictive pre-clinical tolerability testing, it ismandatory for the antibody to exhibit relevant cross-reactivity to atoxicology relevant species, in case of CEACAM6 to non-human primates,preferentially Macaca fascicularis (cynomolgus).

As a prerequisite, a therapeutic antibody needs to bind with highaffinity to human CEACAM6 on cells, to bind selectively to CEACAM6(without binding to any paralogs), to be cross-reactive to monkeyCEACAM6 within one order of magnitude of monovalent K_(D) (to safelyreflect binding on normal tissues in the toxicology monkey model even atlow surface densities under non-avidity based binding conditions), tobind to a similar epitope as on human CEACAM6, to be able to relieveCEACAM6-mediated immunosuppression, to be non-immunogenic in humantherapy (i.e. a human or humanized antibody), and to be stable enough toallow for clinical development, formulation and storage over extendedperiods of time as a pharmaceutical. The latter is important as it hasbeen noted earlier that physical degradation (especially aggregation)may enhance immune response to a therapeutic protein (Hermeling et al.,Pharm Res. 2004 June; 21(6):897-903) and aggregation is closelyconnected to unfolding of IgG and its thermal stability (Vermeer andNorde, Biophys J. 2000 January; 78(1):394-404).

Several anti-CEACAM6 antibodies exist. Most of them are non-humanreagent antibodies, many of them are polyclonal. The specificity andselectivity to human CEACAM6 as well as cross-reactivity to monkeyCEACAM6 is in most of the cases not disclosed or known.

Therapeutic antibodies directed against CEACAM6 are also known in theart. Some are not selective to human CEACAM6 (e.g. MN-3 fromImmunomedics, Neo201/h16C3 from Neogenix; both binding in addition tohuman CEACAM5). A single domain antibody 2A3 and its fusion variants(WO2012040824 and Niu et al., J Control Release. 2012 Jul. 10;161(1):18-24) are not characterized with respect to selectivity andcross-reactivity to monkey CEACAM6.

Selective anti-CEACAM6 antibodies apparently cross-reactive to monkeyCEACAM6 are not disclosed (Strickland et al., J Pathol. 2009 July;218(3):380-90).

The murine antibody 9A6 (Genovac/Aldevron) is the only antibodydescribed to be able to modulate the immunosuppressive activity ofCEACAM6 (Witzens-Harig et al., Blood 2013 May 30; 121(22):4493-503). 9A6inhibits the immunosuppressive activity of CEACAM6, leading to enhancedcytokine secretion by T cells in vitro and anti-tumor efficacy in vivo(Khandelwal et al., Poster Abstract 61, Meeting Abstract from 22ndAnnual International Cancer Immunotherapy Symposium Oct. 6-8, 2014, NewYork City, USA). Although its selectivity appears appropriate, it waspreviously not characterized with regards to its cross-reactivity tomonkey CEACAM6. In addition, its murine nature precludes a directtherapeutic application in humans.

As shown in the examples, the antibody 9A6 binds to recombinant humanCEACAM6 but no binding to recombinant Macaca mulatta or Macacafascicularis CEACAM6 was detected. For comparison, Neo201-hIgG1 was alsotested. This antibody displayed high affinity binding to both human andmonkey CEACAM6. But Neo201 binds to human CEACAM5 and CEACAM6 and istherefore not specific for CEACAM6.

In conclusion there is high need for a therapeutic monoclonal antibodythat comprises the following features:

-   -   i. The antibody is a high affinity binder of human CEACAM6.    -   ii. The antibody is selective to CEACAM6, not binding to any        paralogs, especially CEACAM1, CEACAM3, and CEACAM5.    -   iii. The antibody is cross-reactive to monkey CEACAM6 within one        order of magnitude of monovalent K_(D).    -   iv. The antibody is non-immunogenic in human therapy, i.e. it is        a human or humanized antibody.    -   v. The antibody is able to relieve CEACAM6-mediated        immunosuppression.

Such an antibody does not exist in the prior art. 9A6 binding toN-terminal domain 1 of human CEACAM6 is the only known anti-CEACAM6antibody that is able to relieve CEACAM6-mediated immunosuppression, yetlacks cross-reactivity to monkey CEACAM6 apart from being a mouseantibody. Neo201 binds to a different domain outside of N-terminaldomain 1 of CEACAM6. Therapeutic efficacy of Neo201-hIgG1 has beenpublished to be based on ADCC (Proceedings of the 102nd Annual Meetingof the American Association for Cancer Research; 2011 Apr. 2-6; Orlando,Fla. Philadelphia (PA): AACR: Du et al., Cancer Res Apr. 15, 2011; 71(8Supplement): 4582).

The inventors assumed that relief of CEACAM6-mediated immunosuppressionis connected to binding to N-terminal domain 1. But generation ofantibodies binding to N-terminal domain 1 of CEACAM6 results in achallenging selectivity problem.

The sequence alignment in FIG. 1 shows a very high degree of similarityof protein sequences of human CEACAM6 and human CEACAM3, human CEACAM5and human CEACAM1 throughout the entire extracellular region. The targetregion (domain 1 of human CEACAM6) is especially similar to otherCEACAMs, which is also reflected in Table 7. The paralogs of humanCEACAM6 (e.g. CEACAM1, CEACAM3, and CEACAM5) are much more similar tohuman CEACAM6 than the cynomolgus ortholog. In fact, there are only 2positions in the N-terminal region in the primary sequence that areidentical in human and cynomolgus CEACAM6 but different from amino acidsin the other human paralogs (marked in FIG. 1 with asterisks).

Unexpectedly the inventors were able to find a method to generateantibodies comprising all of the desired selectivity and functionalfeatures.

SUMMARY OF THE INVENTION

This invention is related to antibodies, or antigen-binding antibodyfragments thereof, or variants thereof which display high affinity forhuman and Macaca fascicularis CEACAM6 protein, and which do notsignificantly cross-react with the closely related human CEACAM1, humanCEACAM3, and human CEACAM5. This means the antibodies, orantigen-binding antibody fragments thereof, or variants thereof areselective for CEACAM6. The antibodies provided bind to the N-terminaldomain 1 which is highly conserved among these proteins.

The anti-CEACAM6 antibodies of this invention are able to change invitro the cytokine profile of tumor specific T cells towards a morecytotoxic and/or activated phenotype characterized by increasedIFN-gamma, and/or IL-2 and/or TNF-alpha secretion. Therefore theantibodies of this invention are able to relieve CEACAM6-mediatedimmunosuppression, and induce an immunoactivation, which finally resultsin an anti-tumor efficacy in vivo.

The antibodies of this invention, or antigen-binding antibody fragmentsthereof, or variants thereof interfere with CEACAM6 and CEACAM1interaction which might be a mechanism for the modulation of innate andadaptive immune responses.

The antibodies of the invention are thus suitable for the treatment ofcancer as well as metastases thereof, in particular CEACAM6 expressingtumors, such as colorectal cancer, non-small-cell lung cancer (NSCLC),small cell lung cancer (SCLC), pancreatic cancer, gastric cancer, breastcancer and multiple myeloma.

The invention describes antibodies that are distinguished from existinganti-CEACAM6 antibodies, in that they are able to bind to human andMacaca fascicularis CEACAM6 within one order of magnitude of monovalentK_(D) (to safely reflect binding on normal tissues in toxicology monkeymodel even at low surface densities under non-avidity based bindingconditions) and do not significantly cross-react with the closelyrelated paralogs CEACAM1, CEACAM3, and CEACAM5. So these antibodies aresuitable for preclinical toxicological studies in cynomolgus monkeys toevaluate their safety profiles. As CEACAM6 is not only expressed ontumor cells but also on normal tissues (especially granulocytes but alsoepithelial cells of e.g. lung and gastrointestinal cells—Chan andStanners, Mol Ther. 2004 June; 9(6):775-85; Strickland et al., J Pathol.2009 July; 218(3):380-90), it is absolutely crucial to be able topredict the adverse side effect profile of the therapeutic antibody.This is all the more important, since the anticipated mode of actionwill be inhibition of immunosuppression, i.e. an immunoactivation, whichcan result in serious hazards (incident of CD28 superagonist TGN1412trial), so indirect effects on immune system on top of direct effects ongranulocytes need to be carefully assessed.

Highly preferred anti-CEACAM6 antibodies of the invention are depictedin Table 1 characterized by their structural features.

In some embodiments, the anti-CEACAM6 antibody of the invention binds toan epitope of human CEACAM6, wherein said epitope comprises one or moreamino acid residues selected from the group consisting of Gln60, Asn61,Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 ofSEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibody of the invention bindsto an epitope of human CEACAM6, wherein said epitope comprises the aminoacid residues Gln60, Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90,Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In some embodiments, the anti-CEACAM6 antibody of the inventioninteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises one, two, three four, five, eight, ten, fifteen ormore amino acid residues selected from the group consisting of Pro59,Gln60, Asn61, Arg62, Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88,Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibody of the inventioninteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises the amino acid residues Pro59, Gln60, Asn61, Arg62,Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125,Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

An anti-CEACAM6 antibody of the invention might be co-administered withknown medicaments, and in some instances the antibody might itself bemodified. For example, an antibody could be conjugated to a cytotoxicagent, immunotoxin, toxophore or radioisotope to potentially furtherincrease efficacy.

The invention further provides antibodies which constitute a tool fordiagnosis of malignant or dysplastic conditions in which CEACAM6expression is elevated compared to normal tissue. Provided areanti-CEACAM6 antibodies conjugated to a detectable marker. Preferredmarkers are a radiolabel, an enzyme, a chromophore or a fluorophore.

The invention is also related to polynucleotides encoding the antibodiesof the invention, or antigen-binding fragments thereof, cells expressingthe antibodies of the invention, or antigen-binding fragments thereof,methods for producing the antibodies of the invention, orantigen-binding fragments thereof, methods for inhibiting the growth ofdysplastic cells using the antibodies of the invention, orantigen-binding fragments thereof, and methods for treating anddetecting cancer using the antibodies of the invention, orantigen-binding fragments thereof.

The invention is also related to isolated nucleic acid sequences, eachof which can encode an aforementioned antibody or antigen-bindingfragment thereof that is specific for an epitope of CEACAM6. Nucleicacids of the invention are suitable for recombinant production ofantibodies or antigen-binding antibody fragments. Thus, the inventionalso relates to vectors and host cells containing a nucleic acidsequence of the invention.

Compositions of the invention may be used for therapeutic orprophylactic applications. The invention, therefore, includes apharmaceutical composition comprising an inventive antibody orantigen-binding fragment thereof and a pharmaceutically acceptablecarrier or excipient therefore. In a related aspect, the inventionprovides a method for treating a disorder or condition associated withthe undesired presence of CEACAM6 expressing cells. In a preferredembodiment the aforementioned disorder is cancer. Such method containsthe steps of administering to a subject in need thereof an effectiveamount of the pharmaceutical composition that contains an inventiveantibody as described or contemplated herein.

Further this invention is related to methods to generate this kind ofantibodies. The invention provides instructions for using an antibodylibrary to isolate one or more members of such library that bindsspecifically to CEACAM6. In addition, the invention provides instructionfor immunizing mice to produce hybridoma cell lines that secreteantibodies that bind specifically to CEACAM6 and which arecross-reactive to Macaca fascicularis (cynomolgus monkey) CEACAM6.Instructions for humanization of murine antibodies that bindspecifically to CEACAM6 are also provided by the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: Protein sequence alignment of extracellular regions of humanCEACAM6 paralogs as well as Macaca fascicularis (cynomolgus monkey)CEACAM6 ortholog. Numbers indicate amino acid position after removal ofsignal peptide sequence. Positions in N-terminal region in the primarysequence that are identical in human and cynomolgus CEACAM6 butdifferent from amino acids in this position in the other human paralogsare marked with asterisks. N-terminal domain 1 is boxed.

FIG. 2: Amino acid sequences of the variable domains VL and VH ofTPP-2971. Sequences grafted into human frameworks are highlighted asunderlined bold letters. CDRs according to Kabat definition are writtenas italic letters. Grey shaded letters represent differences of thesequences from TPP-3187 in comparison to TPP-2971.

FIG. 3: Amino acid sequences of the variable domains VL and VH ofTPP-3310 and TPP-3714. Sequences derived from the murine CDRs ofTPP-2971 are highlighted as underlined bold letters. CDRs according toKabat definition are written as italic letters. The antibodies TPP-3310and TPP-3714 differ in two amino acids within the VH frameworkhighlighted as underlined non-bold letters.

FIG. 4: Amino acid sequences of the variable domains VL and VH ofTPP-3820 and TPP-3821. Sequences derived from the murine CDRs ofTPP-3187 are highlighted as underlined bold letters. CDRs according toKabat definition are written as italic letters. The antibodies TPP-3820and TPP-3821 differ in two amino acids within the VH frameworkhighlighted as underlined non-bold letters.

FIG. 5: In vitro pharmacological effect of anti-CEACAM6 antibodies onIFN-gamma secretion of survivin peptide specific T cells and extent ofthis secretion. A+B. IFN-gamma ELISpot assay of survivin-peptidespecific T cells and KS tumor cells. 10,000 KS tumor cells wereco-cultivated together with 2,500 Survivin TC for 20 h. The antibodyconcentration in the co-culture was 30 μg/ml. C. IFN-gamma ELISA assayof survivin-peptide specific TC and KS tumor cells. 10,000 KS tumorcells were co-cultivated together with 20,000 Survivin TC for 20 h. Theantibody concentration in the coculture was 30 μg/ml. X-axis shows thedifferent conditions tested: in A: 1=10,000 KS cells; 2=2,500 T cells;3=no antibody treatment; 4=isotype-matched antibody control; 5=TPP-3470(9A6-hIgG2) 6=TPP-3323; in B: 1=10,000 KS cells; 2=2,500 T cells; 3=noantibody treatment; 4=isotype-matched antibody control; 5=TPP-3470(9A6-hIgG2) 6=TPP-3310; 7=TPP-3707; in C: 1=10,000 KS cells; 2=20,000 Tcells; 3=no antibody treatment; 4=isotype-matched antibody control;5=TPP-3470 (9A6-hIgG2) 6=TPP-3310; 7=TPP-3707; the Y-axis corresponds tothe IFN-gamma spot-counts per well (in A and B) or IFN-gammaconcentration in pg/ml (in C). Asterisks indicate statisticallysignificant results according to Student's t test, unpaired, two-tailed.Error bars represent SEM.

FIG. 6: In vitro pharmacological effect of anti-CEACAM6 antibodies oncytokine secretion (IFN-gamma, IL-2 and TNF-alpha) of survivin peptidespecific T cells. A. IFN-gamma Luminex analysis. B. IL-2 Luminexanalysis. C. TNFa Luminex analsysis. Luminex cytokine analysis ofsurvivin-peptide specific TC and KS tumor cells. 10,000 KS tumor cellswere co-cultivated together with 20,000 Survivin TC for 20 h. Theantibody concentration in the coculture was 30 μg/ml. X-axis depicts thedifferent conditions tested: 1=10,000 KS cells; 2=20,000 T cells; 3=noantibody treatment; 4=isotype-matched antibody control; 5=TPP-3470(9A6-hIgG2) 6=TPP-3310; 7=TPP-3707; the Y-axis corresponds to thecytokine concentration in pg/ml.

FIG. 7: Effect of anti-CEACAM6 antibodies on tumor growth in vivo. 2×10⁶KS breast cancer cells were inoculated s.c. On day 23 and 27tumor-antigen specific T cells (survivin-peptide specific) were injectedi.v. 200 μg of anti-CEACAM6 antibodies or the matched isotype controlwere administered i.p. on day 22, 24, 26 and 28. Tumor growth wasassessed every 2-3 days. Error bars represent SEM. Y-axis=tumor surface(mm²); X-axis=days; TC=survivin-peptide specific T cells. 1=PBS-treated;2 =treatment with T cells and isotype matched antibody control;3=treatment with T cells and TPP-3470 (9A6-hIgG2); 4=treatment with Tcells and TPP-3310; 5=treatment with T cells and TPP-3707.

FIG. 8: Annotated sequences of preferred anti-CEACAM6 antibodies of thisinvention. Provided are protein and DNA sequences for heavy and lightchains of IgGs as well as for VH and VL regions of selected antibodies.Below the sequences important regions are annotated (VH and VL regionsin full length IgGs, and the CDR regions (H-CDR1, H-CDR2, H-CDR3,L-CDR1, L-CDR2, L-CDR3)).

FIG. 9: Cartoon representation of the single N-terminal domain 1 ofhuman CEACAM6 (TPP-1794, white) bound to the Fab fragment APP-1574(heavy and light chains are colored dark and light gray, respectively).

FIG. 10: Details of the protein interface shown in FIG. 9. Selectedresidues are depicted in stick representation and colored as in FIG. 9.The numbering corresponds to TPP-1794 (SEQ-ID NO: 169)

FIG. 11: xCELLigence cytotoxicity assay using survivin-peptide specificCD8⁺ T cells.

A. 20,000 KS breast cancer tumor cells in co-culture with 20,000survivin specific T cells B. 40,000 HCT-116-hC6 tumor cells inco-culture with 20,000 survivin specific T cells. Cytotoxicity wasmonitored for ˜100 h. Antibodies were used at 30 μg/ml finalconcentration: #1, time point of T cell addition; #2, tumor cells only;#3, no antibody; #4, isotype-matched antibody control; #5, anti-PD-L1 Abas human IgG2; #6, TPP-3470 9A6 Ab as human IgG2; #7 TPP-3310 hIgG2.Asterisks indicate statistically significant results according toStudent's t test, unpaired, two-tailed. X, x-axis, Time (Hours); Y,Y-Axis, normalized cell index.

FIG. 12: xCELLigence cytotoxicity assay using patient-derived T cells ofa pancreatic cancer (TIL-12).

A and B: 10,000 HCC2935 tumor cells in co-culture with 50,000 pancreaticcancer infiltrating lymphocyte cells (TIL-12). Cytotoxicity wasmonitored for ˜150 h. An anti-CD3×EpCAM bispecific mAb (0.25 ng/ml) hasbeen added in co-culture to direct T cells against the tumor cellsindependent of HLA.

A: #1,T cell addition; #2 tumor cells only; #3, no antibody; #4,isotype-matched antibody control; #5, anti-PD-L1 Ab as human IgG2; #6,TPP-3470; #7 TPP-3310; Antibodies were used at 30 μg/ml.

B: Concentration dependency of TPP-3310-mediated effect: #1, T celladdition; #2, tumor cells only; #3, TPP-3310 at 0.07 μg/ml; #4, TPP-3310at 0.02 μg/ml; #5, isotype-matched antibody control at 50 μg/ml; #6,TPP-3310 at 0.021 μg/ml; #7, TPP-3310 at 0.062 μg/ml; #8, TPP-3310 at1.85 μg/ml; #9, TPP-3310 at 5.5 μg/ml; #10, TPP-3310 at 16.67 μg/ml;#11, TPP-3310 at 50 μg/ml;

X—x-axis, Time (hours); Y, Y-Axis, normalized cell index

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel antibodies thathave a specific affinity for CEACAM6 and can deliver a therapeuticbenefit to a subject. The antibodies of the invention, which may behuman, humanized or chimeric, can be used in many contexts, which aremore fully described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. The following references, however, canprovide one of skill in the art to which this invention pertains with ageneral definition of many of the terms used in this invention, and canbe referenced and used so long as such definitions are consistent withthe meaning commonly understood in the art. Such references include, butare not limited to, Singleton et al., Dictionary of Microbiology andMolecular Biology (2nd ed. 1994); The Cambridge Dictionary of Scienceand Technology (Walker ed., 1988); Hale & Marham, The Harper CollinsDictionary of Biology (1991); and Lackie et al., The Dictionary of Cell& Molecular Biology (3d ed. 1999); and Cellular and MolecularImmunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. SaundersCompany. Any additional technical resource available to the person ofordinary skill in the art providing definitions of terms used hereinhaving the meaning commonly understood in the art can be consulted. Forthe purposes of the present invention, the following terms are furtherdefined. Additional terms are defined elsewhere in the description. Asused herein and in the appended claims, the singular forms “a,” and“the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a gene” is a reference toone or more genes and includes equivalents thereof known to thoseskilled in the art, and so forth.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

Amino acids may be referred to herein by their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As used herein “CEACAM6” designates the “carcinoembryonicantigen-related cell adhesion molecule 6”, also known as “CD66c”(Cluster of Differentiation 66c), or Non-specific crossreacting antigen,or NCA, or NCA-50/90. CEACAM6 is a glycosylphosphatidylinositol(GPI)-linked cell surface protein involved in cell-cell adhesion.CEACAM6 is highly expressed on the surface of different tumor cells likecolon, pancreatic, breast and lung cancer.

A reference sequence for human CEACAM6 is available fromUniProtKB/Swiss-Prot data base under accession number P40199.3 (SEQ-IDNO:179=TPP-4639), including signal peptide (positions 1-34) andpropetide chain (positions 321-344). A single nucleotide polymorphismhas been observed at position 239 (G to V exchange). The matureextracellular domain of human CEACAM6 consists of amino acids atposition 35-320 of SEQ-ID No: 179.

human CEACAM6 (SEQ-ID NO: 179)MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWVNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDGPTISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQAHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI 

A Macaca fascicularis (cynomolgus monkey) protein sequence of CEACAM6was deduced by the inventors and is represented by TPP-4189 (SEQ-ID No:177). The mature extracellular domain of cynomolgus CEACAM6 consists ofamino acids at position 35-320 of SEQ-ID No: 177.

Macaca fascicularis (cynomolgus monkey) CEACAM6 (SEQ-ID NO: 177)MGPPSAPPCRICVPWKEVLLTASLLTFWSPPTTAQLTIESRPFNVAEGKEVLLLAHNLPQNTLGFNWYKGERVDAKRLIVAYVIGTQQTTPGPAHSGREMIYSNASLLIQNVTQNDTGSYTLQAIKEDLVTEEATGRFWVYPELPKPYITSNNSNPVEDKDAVDFTCEPDIHSTTYLWWVNDQSLPVSPRLQLSNGNRTLTLLSVKRNDAGAYECEIQNPVSANLSDPVILNVLYGPDVPTISPSNSNYRPGENLNLSCHAASNPTAQYSWFVNGTFQQSTQELFIPNITVNNSGSYMCQAYNSATGLNRTTVMMITVSGSAPGLSAVATVGIMIGVLARVALI 

Domain organization of human and Macaca fascicularis (cynomolgus monkey)CEACAM6 is as follows (based on UniProtKB/Swiss-Prot data base sequenceunder accession number P40199.3 and SEQ-ID NO:179=TPP-4639 & SEQ-ID No:177=TPP-4189, respectively):

Positions on SEQ-ID NO: 179 = TPP-4639 Human and cynomolgus CEACAM6 andSEQ-ID No: 177 = TPP-4189, domains respectively Domain 1  35-142 alsoknown as N domain also known as N-terminal domain 1 (Ig-like V-type)Domain 2 145-232 also known as A domain (Ig-like C2-type 1) Domain 3237-314 also known as B domain (Ig-like C2-type 2)

human CEACAM1 full-length protein is available from UniProtKB/Swiss-Protdata base under accession number P13688.2 (SEQ-ID No: 173=TPP-4185). Themature extracellular domain of human CEACAM1 consists of amino acids atposition 35-428 of SEQ-ID No: 173.

human CEACAM1 (SEQ-ID NO: 173)MGHLSAPLHRVRVPWQGLLLTASLLTFWPIPPTTAQLTTESMPFITVAEGKEVLLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQSLPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDTYYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCNRTTVKTIIVTELSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLSQGNTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENGLSPGAIAGIVIGVVALVALIAVALACFLHFGKTGRASDQRDLTEHKPSVSNHTQDHSNDPPNKMNEVTYSTLNFEAQQPTQPTSASPSLTATEIIYSEVKKQ 

human CEACAM3 full-length protein is available from UniProtKB/Swiss-Protdata base under accession number P40198.2 (SEQ-ID No: 175=TPP-4187). Themature extracellular domain of human CEACAM3 consists of amino acids atposition 35-155 of SEQ-ID No: 175.

human CEACAM3 (SEQ-ID NO: 175)MGPPSASPHRECIPWQGLLLTASLLNFWNPPTTAKLTIESMPLSVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNSLIVGYVIGTQQATPGAAYSGRETTYTNASLLIQNVTQNDIGFYTLQVIKSDLVNEEATGQFHVYQENAPGLPVGAVAGIVTGVLVGVALVAALVCFLLLAKTGRTSIQRDLKEQQPQALAPGRGPSHSSAFSMSPLSTAQAPLPNPRTAASIYEELLKHDTNIYCRMDHKAEV AS 

human CEACAM5 full-length protein is available from UniProtKB/Swiss-Protdata base under accession number P06731.3 (SEQ-ID No: 176=TPP-4188). Themature extracellular domain of human CEACAM5 consists of amino acids atposition 35-685 of SEQ-ID No: 176.

human CEACAM5 (SEQ-ID NO: 176)MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVESITVSASGTSPGLSAGATVGIMIGVLVGVA LI 

The terms “anti-CEACAM6 antibody” and “an antibody that binds toCEACAM6” refer to an antibody that is capable of binding CEACAM6 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting CEACAM6. In one embodiment, theextent of binding of an anti-CEACAM6 antibody to an unrelated,non-CEACAM6 protein is less than about 5%, or preferably less than about2% of the binding of the antibody to CEACAM6 as measured, e.g., by asurface plasmon resonance (SPR). In certain embodiments, an antibodythat binds to CEACAM6 has a dissociation constant (K_(D)) of ≤1 μM, ≤100nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-CEACAM6 antibody binds to an epitope of CEACAM6that is conserved among CEACAM6 from different species.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules, preferably comprised of four polypeptidechains, two heavy (H) chains and two light (L) chains which aretypically inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region cancomprise e.g. three domains CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain (CL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is typicallycomposed of three CDRs and up to four FRs arranged from amino-terminusto carboxy-terminus e.g. in the following order: FR1, CDR1, FR2, CDR2,FR3, CDR3, FR4.

As used herein, the term “Complementarity Determining Regions” (CDRs;e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (e.g. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; (Kabat et al.,Sequences of Proteins of Immulological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26- 32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these maybe further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. A preferredclass of immunoglobulins for use in the present invention is IgG.

The heavy-chain constant domains that correspond to the differentclasses of antibodies are called [alpha], [delta], [epsilon], [gamma],and [mu], respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.As used herein antibodies are conventionally known antibodies andfunctional fragments thereof.

A “functional fragment” or “antigen-binding antibody fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hyper variable region(s) of anantibody, e.g., the CDR1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320).

“Functional fragments”, “antigen-binding antibody fragments”, or“antibody fragments” of the invention include but are not limited toFab, Fab′, Fab′-SH, F(ab′)₂, and Fv fragments; diabodies; single domainantibodies (DAbs), linear antibodies; single-chain antibody molecules(scFv); and multispecific, such as bi- and tri-specific, antibodiesformed from antibody fragments (C. A. K Borrebaeck, editor (1995)Antibody Engineering (Breakthroughs in Molecular Biology), OxfordUniversity Press; R. Kontermann & S. Duebel, editors (2001) AntibodyEngineering (Springer Laboratory Manual), Springer Verlag). An antibodyother than a “multi-specific” or “multi-functional” antibody isunderstood to have each of its binding sites identical. The F(ab′)₂ orFab may be engineered to minimize or completely remove theintermolecular disulfide interactions that occur between the CH1 and CLdomains.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

Variants of the antibodies or antigen-binding antibody fragmentscontemplated in the invention are molecules in which the bindingactivity of the antibody or antigen-binding antibody fragment ismaintained.

“Binding proteins” contemplated in the invention are for exampleantibody mimetics, such as Affibodies, Adnectins, Anticalins, DARPins,Avimers, Nanobodies (reviewed by Gebauer M. et al., Curr. Opinion inChem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion inPharmacology 2008; 8:608-617).

A “human” antibody or antigen-binding fragment thereof is hereby definedas one that is not chimeric (e.g., not “humanized”) and not from (eitherin whole or in part) a non-human species. A human antibody orantigen-binding fragment thereof can be derived from a human or can be asynthetic human antibody. A “synthetic human antibody” is defined hereinas an antibody having a sequence derived, in whole or in part, in silicofrom synthetic sequences that are based on the analysis of known humanantibody sequences. In silico design of a human antibody sequence orfragment thereof can be achieved, for example, by analyzing a databaseof human antibody or antibody fragment sequences and devising apolypeptide sequence utilizing the data obtained there from. Anotherexample of a human antibody or antigen-binding fragment thereof is onethat is encoded by a nucleic acid isolated from a library of antibodysequences of human origin (e.g., such library being based on antibodiestaken from a human natural source). Examples of human antibodies includeantibodies as described in Söderlind et al., Nature Biotech. 2000,18:853-856.

A “humanized antibody” or humanized antigen-binding fragment thereof isdefined herein as one that is (i) derived from a non-human source (e.g.,a transgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; (ii) where amino acidsof the framework regions of a non-human antibody are partially exchangedto human amino acid sequences by genetic engineering or (iii)CDR-grafted, wherein the CDRs of the variable domain are from anon-human origin, while one or more frameworks of the variable domainare of human origin and the constant domain (if any) is of human origin.

A “chimeric antibody” or antigen-binding fragment thereof is definedherein as one, wherein the variable domains are derived from a non-humanorigin and some or all constant domains are derived from a human origin.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the term “monoclonal” indicatesthe character of the antibody as not being a mixture of discreteantibodies. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins. The term “monoclonal” is not to be construedas to require production of the antibody by any particular method. Theterm monoclonal antibody specifically includes chimeric, humanized andhuman antibodies.

An “isolated” antibody is one that has been identified and separatedfrom a component of the cell that expressed it. Contaminant componentsof the cell are materials that would interfere with diagnostic ortherapeutic uses of the antibody, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes.

An “isolated” nucleic acid is one that has been identified and separatedfrom a component of its natural environment. An isolated nucleic acidincludes a nucleic acid molecule contained in cells that ordinarilycontain the nucleic acid molecule, but the nucleic acid molecule ispresent extrachromosomally or at a chromosomal location that isdifferent from its natural chromosomal location.

As used herein, an antibody “binds specifically to”, is “specificto/for” or “specifically recognizes” an antigen of interest, e.g. atumor-associated polypeptide antigen target, is one that binds theantigen with sufficient affinity such that the antibody is useful as atherapeutic agent in targeting a cell or tissue expressing the antigen,and does not significantly cross-react with other proteins or does notsignificantly cross-react with proteins other than orthologs andvariants (e.g. mutant forms, splice variants, or proteolyticallytruncated forms) of the aforementioned antigen target. The term“specifically recognizes” or “binds specifically to” or is “specificto/for” a particular polypeptide or an epitope on a particularpolypeptide target as used herein can be exhibited, for example, by anantibody, or antigen-binding fragment thereof, having a monovalent K_(D)for the antigen of less than about 10⁻⁴ M, alternatively less than about10⁻⁵ M, alternatively less than about 10⁻⁶ M, alternatively less thanabout 10⁻⁷ M, alternatively less than about 10⁻⁸ M, alternatively lessthan about 10⁻⁹ M, alternatively less than about 10⁻¹⁰ M, alternativelyless than about 10⁻¹¹ M, alternatively less than about 10⁻¹² M, or less.An antibody “binds specifically to,” is “specific to/for” or“specifically recognizes” an antigen if such antibody is able todiscriminate between such antigen and one or more reference antigen(s).In its most general form, “specific binding”, “binds specifically to”,is “specific to/for” or “specifically recognizes” is referring to theability of the antibody to discriminate between the antigen of interestand an unrelated antigen, as determined, for example, in accordance withone of the following methods. Such methods comprise, but are not limitedto surface plasmon resonance (SPR), Western blots, ELISA-, RIA-, ECL-,IRMA-tests and peptide scans. For example, a standard ELISA assay can becarried out. The scoring may be carried out by standard colordevelopment (e.g. secondary antibody with horseradish peroxidase andtetramethyl benzidine with hydrogen peroxide). The reaction in certainwells is scored by the optical density, for example, at 450 nm. Typicalbackground (=negative reaction) may be 0.1 OD; typical positive reactionmay be 1 OD. This means the difference positive/negative is more than5-fold, 10-fold, 50-fold, and preferably more than 100-fold. Typically,determination of binding specificity is performed by using not a singlereference antigen, but a set of about three to five unrelated antigens,such as milk powder, BSA, transferrin or the like.

“Binding affinity” or “affinity” refers to the strength of the total sumof non-covalent interactions between a single binding site of a moleculeand its binding partner. Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair (e.g. an antibody andan antigen). The dissociation constant “K_(D)” is commonly used todescribe the affinity between a molecule (such as an antibody) and itsbinding partner (such as an antigen) i.e. how tightly a ligand binds toa particular protein. Ligand-protein affinities are influenced bynon-covalent intermolecular interactions between the two molecules.Affinity can be measured by common methods known in the art, includingthose described herein. In one embodiment, the “K_(D)” or “K_(D) value”according to this invention is measured by using surface plasmonresonance assays using suitable devices including but not limited toBiacore instruments like Biacore T100, Biacore T200, Biacore 2000,Biacore 4000, a Biacore 3000 (GE Healthcare Biacore, Inc.), or a PrateOnXPR36 instrument (Bio-Rad Laboratories, Inc.).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains, orcombinations thereof and usually have specific three dimensionalstructural characteristics, as well as specific charge characteristics.

An “antibody that binds to the same epitope” as a reference antibody or“an antibody which competes for binding” to a reference antibody refersto an antibody that blocks binding of the reference antibody to itsantigen in a competition assay by 10%, 20%, 30%, 40%, 50% or more, andconversely, the reference antibody blocks binding of the antibody to itsantigen in a competition assay by 10%, 20%, 30%, 40%, 50% or more. Anexemplary competition assay is provided herein.

An “antibody which binds to an epitope of a target protein Z whereinsaid epitope comprises the amino acid residues X1, X2, X3, . . . ” is anantibody which comprises atoms within 5 Å, preferentially within 4 Å, toatoms of said amino acid residues X1, X2, X3, . . . of the targetprotein Z after binding of the antibody to its target protein. Suchepitopes can be determined by using an X-ray crystal structure asexemplified in example 16.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc gamma receptors(FcγRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils,and macrophages) enable these cytotoxic effector cells to bindspecifically to an antigen-bearing target cell and subsequently kill thetarget cell e.g. with cytotoxins. To assess ADCC activity of an antibodyof interest, an in vitro ADCC assay, such as that described in U.S. Pat.Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may beperformed. Useful effector cells for such assays include PBMC and NKcells.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding aredescribed, e.g., in U.S. Pat. No. 6,194,551 BI and WO 1999/51642.

As used herein, a “naked antibody” refers to an antibody that is notconjugated to a heterologous moiety (e.g. a cytotoxic moiety) orradiolabel. This naked antibody may be present in a pharmaceuticalcomposition.

The term “immunoconjugate” (interchangeably referred to as“antibody-drug conjugate,” or “ADC”) refers to an antibody conjugated toone or more cytotoxic or cytostatic agents, such as a chemotherapeuticagent, a drug, a growth inhibitory agent, a toxin (e.g., a proteintoxin, an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Immunoconjugates have been used for the local deliveryof cytotoxic agents, i.e., drugs that kill or inhibit the growth orproliferation of cells, in the treatment of cancer (e.g. Liu et al.,Proc Natl. Acad. Sci. (1996), 93, 8618-8623)). Immunoconjugates allowfor the targeted delivery of a drug moiety to a tumor, and intracellularaccumulation therein, where systemic administration of unconjugateddrugs may result in unacceptable levels of toxicity to normal cellsand/or tissues. Toxins used in antibody-toxin conjugates includebacterial toxins such as diphtheria toxin, plant toxins such as ricin,small molecule toxins such as geldanamycin. The toxins may exert theircytotoxic effects by mechanisms including tubulin binding, DNA binding,or topoisomerase inhibition.

“Percent (%) sequence identity” with respect to a referencepolynucleotide or polypeptide sequence, respectively, is defined as thepercentage of nucleic acid or amino acid residues, respectively, in acandidate sequence that are identical with the nucleic acid or aminoacid residues, respectively, in the reference polynucleotide orpolypeptide sequence, respectively, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Conservative substitutions are not considered as part of thesequence identity. Preferred are un-gapped alignments. Alignment forpurposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

“Sequence homology” indicates the percentage of amino acids that eitheris identical or that represent conservative amino acid substitutions.

The term “maturated antibodies” or “maturated antigen-binding fragments”such as maturated Fab variants includes derivatives of an antibody orantibody fragment exhibiting stronger binding—i. e. binding withincreased affinity—to a given antigen such as the extracellular domainof a target protein. Maturation is the process of identifying a smallnumber of mutations e.g. within the six CDRs of an antibody or antibodyfragment leading to this affinity increase. The maturation process isthe combination of molecular biology methods for introduction ofmutations into the antibody and screening for identifying the improvedbinders.

An “antagonistic” antibody or a “blocking” antibody is one whichsignificantly inhibits (either partially or completely) a biologicalactivity of the antigen it binds.

An “agonistic” antibody or an antibody with “agonistic activity” is onethat binds to its target and induces the activation (either partially orcompletely) of the respective target, that e.g. leads to activation ofthe signaling pathways or biological effects (either partially orcompletely) that are mediated by the respective target. An “agonistic”antibody or an antibody with “agonistic activity” as used herein is anantibody which may mimic at least one of the functional activities of apolypeptide of interest.

The term “pharmaceutical formulation”/“pharmaceutical composition”refers to a preparation which is in such form as to permit thebiological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants”, “transformed cells”, “transfectants”,“transfected cells”, and “transduced cells”, which include the primarytransformed/transfected/transduced cell and progeny derived therefromwithout regard to the number of passages. Progeny may not be completelyidentical in nucleic acid content to a parent cell, but may containmutations. Mutant progeny that have the same function or biologicalactivity as screened or selected for in the originally transformed cellare included herein.

Antibodies of this Invention

This invention is related to antibodies, or antigen-binding antibodyfragments thereof, or variants thereof which bind specifically to humanCEACAM6 and Macaca fascicularis CEACAM6, and which therefore do notsignificantly cross-react with the closely related human CEACAM1, humanCEACAM3, and human CEACAM5.

Antibodies of this invention, or antigen-binding antibody fragmentsthereof, or variants thereof bind specifically to the matureextracellular domain of CEACAM6 and the mature extracellular domain ofMacaca fascicularis CEACAM6, and do not significantly cross-react withthe closely related mature extracellular domains of human CEACAM1, ofhuman CEACAM3, and of human CEACAM5. The mature extracellular domainsmight be part of the full length proteins expressed on the cell surfaceas well as soluble proteins (naturally occurring or recombinantlyexpressed).

Antibodies of this invention, or antigen-binding antibody fragmentsthereof, or variants thereof bind specifically to proteins comprisingthe mature extracellular domain of human CEACAM6 and/or the matureextracellular domain of Macaca fascicularis CEACAM6, and do notsignificantly cross-react with proteins comprising only the closelyrelated mature extracellular domains of human CEACAM1 and/or of humanCEACAM3, and/or of human CEACAM5.

It is an embodiment of this invention to provide antibodies orantigen-binding antibody fragments thereof, or variants thereof whichare specific for human and Macaca fascilularis CEACAM6, which means, theantibodies are cross-reactive to human and Macaca fascilularis CEACAM6.

It is an embodiment of this invention to provide antibodies orantigen-binding antibody fragments thereof, or a variants thereof whichare selective for CEACAM6, which means these do not significantlycross-react with the closely related CEACAM1, CEACAM3, and CEACAM5.

It is another embodiment of the invention to provide antibodies, orantigen-binding antibody fragments thereof, or variants thereof, whichbind to human CEACAM6 and are cross-reactive to CEACAM6 of anotherspecies including, but not limited to, monkey with similar affinity.Preferably, said other species is a non-human primate, such as forexample Macaca fascicularis, Macaca mulatta, orang-utang, gorilla, andchimpanzee. Most preferably, the antibodies, or antigen-binding antibodyfragments thereof, or variants thereof bind to human CEACAM6 and arecross-reactive to cynomolgus CEACAM6.

A monoclonal antibody binding to antigen 1 (Ag1) is “cross-reactive” toantigen 2 (Ag2) when the EC50 and/or K_(D) values are in a similar rangefor both antigens. In the present disclosure, a monoclonal antibodybinding to Ag1 is cross-reactive to Ag2 when the ratio of affinity forAg 1 to affinity for Ag2 is equal or less 10 (≤10) and equal or greaterthan 0.1 (≥0.1), which means that the affinities for Ag1 and Ag2 do notdiffer more than a factor of 10 (the affinities are within one order ofmagnitude of monovalent K_(D)), on condition that affinities aremeasured with the same method in the same experimental setting for bothantigens.

Accordingly, the antibody according to the invention has a ratio ofaffinity for human CEACAM6 to the affinity for Macaca fascicularisCEACAM6 which is equal or less 10 (≥10) and equal or greater than 0.1(≥0.1), which means that the affinities for human and Macacafascicularis CEACAM6 do not differ more than a factor of 10 (theaffinities are within one order of magnitude of monovalent K_(D)). Thus,the antibody of this invention, or antigen-binding antibody fragmentthereof, or variant thereof according to the invention may be used intoxicological studies performed in monkeys because the toxicity profileobserved in monkeys would be relevant to anticipate potential adverseeffects in humans.

A monoclonal antibody binding to antigen 1 (Ag1) is “not significantlycross-reactive” to antigen 3 (Ag3) when the affinities are verydifferent for the two antigens. Affinity for Ag3 may not be measurableif the binding response (the measured binding signal in an assay) is toolow. In the present application, a monoclonal antibody binding to Ag1 is“not significantly cross-reactive” to Ag3, when the binding response(the measured binding signal in an assay) of the monoclonal antibody toAg3 is less than 5%, preferably less than 2% of the binding response ofthe same monoclonal antibody to Ag1 in the same experimental setting andat the same antibody concentration. In practice, the antibodyconcentration used can be the EC₅₀ or K_(D) or the concentrationrequired to reach the saturation plateau obtained with Ag1.

According to the invention the antibodies or antigen-binding antibodyfragments thereof, or variants thereof do not significantly cross reactwith human CEACAM1, human CEACAM3, and human CEACAM5.

A preferred embodiment of the invention has an affinity for humanCEACAM6 and Macaca fascicularis CEACAM6 which is 5400 nM, preferably≤200 nM, alternatively preferably ≤100 nM, determined as monovalentaffinity to recombinant CEACAM6 (see Example 2) as shown in Table 13,Table 18 and Table 20.

According to the invention the antibodies or antigen-binding antibodyfragments thereof, or variants thereof bind to human CEACAM6 domain 1(represented by amino acids 35-142 of SEQ-ID NO:179) and to Macacafascicularis CEACAM6 domain 1 (represented by amino acids 35-142 ofSEQ-ID NO:177) (affinities are within one order of magnitude ofmonovalent KO. The CEACAM6 domain 1 is also known as N domain.

It is an embodiment of the invention to provide antibodies orantigen-binding antibody fragments thereof, or variants thereof whichbind specifically to human CEACAM6 domain 1 (represented by amino acids35-142 of SEQ-ID NO:179) and specifically to Macaca fascicularis CEACAM6domain 1 (represented by amino acids 35-142 of SEQ-ID NO:177) withaffinities (monovalent K_(D)) which are within one order of magnitude.Highly preferred are antibodies or antigen-binding antibody fragmentsthereof, or variants thereof which have an affinity for both, humanCEACAM6 domain 1 and Macaca fascicularis CEACAM6 domain 1 of K_(D)≤100nM.

Antibodies of this invention, or antigen-binding antibody fragmentsthereof, or variants thereof bind specifically to proteins comprisinghuman CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-IDNO:179) and/or Macaca fascicularis CEACAM6 domain 1 (represented byamino acids 35-142 of SEQ-ID NO:177).

Antibodies of this invention, or antigen-binding antibody fragmentsthereof, or variants thereof bind specifically to proteins comprisinghuman CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-IDNO:179) and/or Macaca fascicularis CEACAM6 domain 1 (represented byamino acids 35-142 of SEQ-ID NO:177) and do not significantlycross-react with the closely related human CEACAM1, human CEACAM3, andhuman CEACAM5.

Antibodies of this invention, or antigen-binding antibody fragmentsthereof, or variants thereof bind specifically to proteins comprisinghuman CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-IDNO:179) and/or Macaca fascicularis CEACAM6 domain 1 (represented byamino acids 35-142 of SEQ-ID NO:177) and do not significantlycross-react with proteins comprising only the closely related matureextracellular domains of human CEACAM1, and/or of human CEACAM3, and/orof human CEACAM5.

It is an embodiment of the invention to provide antibodies, orantigen-binding antibody fragments thereof, or variants thereof whichbind specifically to the mature extracellular domain of CEACAM6 and themature extracellular domain of Macaca fascicularis CEACAM6, and competefor binding to the 9A6 antibody (Genovac/Aldevron) on human CEACAM6 anddo not significantly cross-react with the closely related matureextracellular domains of human CEACAM1, of human CEACAM3, and of humanCEACAM5.

It is an embodiment of the invention to provide antibodies, orantigen-binding antibody fragments thereof, or a variants thereof whichbind specifically to human CEACAM6 domain 1 (represented by amino acids35-142 of SEQ-ID NO:179) and to Macaca fascicularis CEACAM6 domain 1(represented by amino acids 35-142 of SEQ-ID NO:177), and compete forbinding to the 9A6 antibody (Genovac/Aldevron) on human CEACAM6.

It is an embodiment of the invention to provide antibodies orantigen-binding antibody fragments thereof, or variants thereof whichbind specifically to human CEACAM6 and Macaca fascicularis CEACAM6, andwhich interfere with CEACAM6 and CEACAM1 interaction. An antibodyinterferes with the CEACAM6 and CEACAM1 interaction, when the bindingsignal of a preformed antibody-CEACAM6-complex is more than 20%,preferably more than 50% reduced compared to that of the CEACAM6 proteinalone in a typical binding assay with CEACAM1 which is provided in theexamples. Interference with CEACAM6 and CEACAM1 interaction might be amechanism for the modulation of innate and adaptive immune responses.

It is an embodiment of the invention to provide antibodies orantigen-binding antibody fragments thereof, or variants thereof whichbind specifically to human CEACAM6 and Macaca fascicularis CEACAM6, andwhich have an immunomodulatory activity. “Immunomodulation” is theadjustment of the immune response to a desired level as inimmunopotention, immunosuppression or induction of immunologictolerance. An “immunomodulatory antibody” drug is an immuneresponse-modifying agent that stimulates or suppresses the immuneresponse for the treatment of a disease e.g. cancer oranti-inflammation. The invention provides an anti-CEACAM6immunomodulatory antibody, as shown in Example 11, which blocks CEACAM6which is an immune cell suppressive ligand on cancer cells andpotentially other cells including components of the immune system whichresults into a change of the immune marker expression profile andactivation status of T cells towards an anti-tumor T cell re-activationand an effective anti-cancer immune response. An antibody binding toCEACAM6 domain 1 is preferred.

It is an embodiment of the invention to provide antibodies orantigen-binding antibody fragments thereof, or variants thereof whichbind specifically to human CEACAM6 and Macaca fascicularis CEACAM6, andwhich are able to relieve CEACAM6 mediated immunosuppression of tumorantigen specific T cells as measured by either IFN-gamma secretion oftumor antigen specific T cells or the number of IFN-gamma secretingactivated T cells. An anti-CEACAM6 antibody is able to relieve CEACAM6mediated immunosuppression of tumor antigen specific T cells if anantibody in the co-culture of tumor specific T cells with tumor cellsyields a >1.2, preferably >1.5 times increase in IFN-gamma secretioncompared to the control samples. Preferably the tumor antigen specific Tcells are CD8⁺ T cells. Preferred is an antibody which does notsignificantly cross-react with the closely related CEACAM1, CEACAM3, andCEACAM5 and which binds to CEACAM6 domain 1. This is exemplary shown byblockade of CEACAM6 by antibodies of the invention in the co-culture ofsurvivin-peptide specific CD8⁺ T cells with KS tumor cells, which yieldsa >1.5 times increase in IFN-gamma secretion compared to the controlsamples that were treated with the isotype-matched control (FIG. 5 andFIG. 6).

It is an embodiment of the invention to provide antibodies orantigen-binding antibody fragments thereof, or variants thereof whichbind specifically to human CEACAM6 and Macaca fascicularis CEACAM6, andwhich are able to change the cytokine profile of tumor antigen specificT cells towards a more cytotoxic and/or activated phenotypecharacterized by increased IFN-gamma and/or IL-2 and/or TNF-alphasecretion. Preferably the tumor antigen specific T cells are CD8⁺ Tcells and the phenotype is an increased IFN-gamma and IL-2 and TNF-alphasecretion. An anti-CEACAM6 antibody is able to change the cytokineprofile of tumor antigen specific T cells towards a more cytotoxicand/or activated phenotype if the antibody in the co-culture of tumorspecific CD8⁺ T cells with tumor cells yields a >1.2, preferably >1.5times increase in IFN-gamma and/or IL-2 and/or TNF-alpha secretioncompared to the control samples that were treated with theisotype-matched control. Blockade of CEACAM6 by antibodies of theinvention in the co-culture of survivin-peptide specific T cells with KStumor cells yields a >1.5 times increase in IFN-gamma, IL-2 andTNF-alpha secretion compared to the control samples that were treatedwith the isotype-matched control (FIG. 6).

It is an embodiment of the invention to provide antibodies, orantigen-binding antibody fragments thereof, or variants thereof, whichbind to a broad range of different CEACAM6 expressing cell linesincluding, but not limited to the ones shown in the examples. Theseexamples include human cell lines from many tumor origins (e.g. NSCLC,SCLC, CRC, PancCA, BreastCA, GastricCA, multiple myeloma, cervix, skincancer which represent cancer indications previously described in theliterature to be CEACAM6 positive (see introduction or review Beauchimenand Arabzadeh, Cancer Metastasis Rev. 2013 December; 32(3-4):643-71).

It is an embodiment of the invention to provide antibodies, orantigen-binding antibody fragments thereof, or variants thereof that aresafe for human administration. Preferably the antibodies are chimeric,humanized or human. Highly preferred are humanized or human antibodies.

Nevertheless in certain assays an expression of the antibodies of thisinvention as murine IgG is preferred; immunohistochemistry with humansamples for example can be analyzed more easily by using murineantibodies or human-mouse chimeric antibodies.

It is another embodiment of the invention to provide antibodies whichconstitute a tool for diagnosis of malignant or dysplastic conditions inwhich CEACAM6 expression is elevated compared to normal tissue or whereCEACAM6 is shed from the cell surface and becoming detectable in serum.Provided are anti-CEACAM6 antibodies conjugated to a detectable marker.Preferred markers are a radiolabel, an enzyme, a chromophore or afluorophore.

Throughout this document, reference is made to the following preferredanti-CEACAM6 antibodies of the invention as depicted in Table 1.

TABLE 1 Protein sequences of preferred antibodies of this invention SEQID SEQ ID SEQ ID SEQ ID NO: NO: NO: SEQ ID SEQ ID SEQ ID NO: SEQ ID SEQID SEQ ID IgG IgG VH NO: NO: NO: VL NO: NO: NO: Heavy Light ProteinH-CDR1 H-CDR2 H-CDR3 Protein L-CDR1 L-CDR2 L-CDR3 Chain Chain TPP-2971mIgG1 3 4 5 6 7 8 9 10 TPP-3308 hIgG2 - 33 34 35 36 37 38 39 40 43 44chim TPP-3310 hIgG2 47 48 49 50 51 52 53 54 57 58 TPP-3714 hIgG2 119 120121 122 123 124 125 126 129 130 TPP-3187 mIgG1 23 24 25 26 27 28 29 30TPP-3323 hIgG2 - 75 76 77 78 79 80 81 82 85 86 chim TPP-3820 hIgG2 133134 135 136 137 138 139 140 143 144 TPP-3821 hIgG2 147 148 149 150 151152 153 154 157 158 TPP-3186 mIgG1 13 14 15 16 17 18 19 20 TPP-3322hIgG2 - 61 62 63 64 65 66 67 68 71 72 chim TPP-3707 hIgG2 105 106 107108 109 110 111 112 115 116 TPP-3705 hIgG2 91 92 93 94 95 96 97 98 101102

The sequences of preferred antibodies of this invention orantigen-binding fragments thereof depicted in Table 1 are furtherprovided and explained in FIG. 8.

TPP-3308 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 43 and a light chain region corresponding toSEQ ID NO: 44.

TPP-3310 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 57 and a light chain region corresponding toSEQ ID NO: 58.

TPP-3714 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 129 and a light chain region correspondingto SEQ ID NO: 130.

TPP-3323 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 85 and a light chain region corresponding toSEQ ID NO: 86.

TPP-3820 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 143 and a light chain region correspondingto SEQ ID NO: 144.

TPP-3821 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 157 and a light chain region correspondingto SEQ ID NO: 158.

TPP-3322 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 71 and a light chain region corresponding toSEQ ID NO: 72.

TPP-3707 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 115 and a light chain region correspondingto SEQ ID NO: 116.

TPP-3705 represents an antibody comprising a heavy chain regioncorresponding to SEQ ID NO: 101 and a light chain region correspondingto SEQ ID NO: 102.

In a further preferred embodiment the antibodies or antigen-bindingfragments comprise heavy or light chain CDR sequences which are at least50%, 55%, 60% 70%, 80%, 90, or 95% identical to at least one, preferablycorresponding, CDR sequence of the antibodies “TPP-2971”, “TPP-3186”,“TPP-3187”, “TPP-3308”, “TPP-3310”, “TPP-3322”, “TPP-3323”, “TPP-3705”,“TPP-3707”, “TPP-3714”, “TPP-3820”, “TPP-3821” or at least 50%, 60%,70%, 80%, 90%, 92% or 95% identical to the VH or VL sequence of“TPP-2971”, “TPP-3186”, “TPP-3187”, “TPP-3308”, “TPP-3310”, “TPP-3322”,“TPP-3323”, “TPP-3705”, “TPP-3707”, “TPP-3714”, “TPP-3820”, “TPP-3821”,respectively.

In a further preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises at least one CDR sequence orat least one variable heavy chain or variable light chain sequence asdepicted in Table 1.

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:4 (H-CDR1), SEQ ID NO:5 (H-CDR2) and SEQID NO:6 (H-CDR3) and comprises a light chain antigen-binding region thatcomprises SEQ ID NO:8 (L-CDR1), SEQ ID NO:9 (L-CDR2) and SEQ ID NO:10(L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:34 (H-CDR1), SEQ ID NO:35 (H-CDR2) andSEQ ID NO:36 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:38 (L-CDR1), SEQ ID NO:39 (L-CDR2) and SEQ IDNO:40 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:48 (H-CDR1), SEQ ID NO:49 (H-CDR2) andSEQ ID NO:50 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:52 (L-CDR1), SEQ ID NO:53 (L-CDR2) and SEQ IDNO:54 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:120 (H-CDR1), SEQ ID NO:121 (H-CDR2) andSEQ ID NO:122 (H-CDR3) and comprises a light chain antigen-bindingregion that comprises SEQ ID NO:124 (L-CDR1), SEQ ID NO:125 (L-CDR2) andSEQ ID NO:126 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:24 (H-CDR1), SEQ ID NO:25 (H-CDR2) andSEQ ID NO:26 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:28 (L-CDR1), SEQ ID NO:29 (L-CDR2) and SEQ IDNO:30 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:76 (H-CDR1), SEQ ID NO:77 (H-CDR2) andSEQ ID NO:78 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:80 (L-CDR1), SEQ ID NO:81 (L-CDR2) and SEQ IDNO:82 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:134 (H-CDR1), SEQ ID NO:135 (H-CDR2) andSEQ ID NO:136 (H-CDR3) and comprises a light chain antigen-bindingregion that comprises SEQ ID NO:138 (L-CDR1), SEQ ID NO:139 (L-CDR2) andSEQ ID NO:140 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:148 (H-CDR1), SEQ ID NO:149 (H-CDR2) andSEQ ID NO:150 (H-CDR3) and comprises a light chain antigen-bindingregion that comprises SEQ ID NO:152 (L-CDR1), SEQ ID NO:153 (L-CDR2) andSEQ ID NO:154 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:62 (H-CDR1), SEQ ID NO:63 (H-CDR2) andSEQ ID NO:64 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:66 (L-CDR1), SEQ ID NO:67 (L-CDR2) and SEQ IDNO:68 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:14 (H-CDR1), SEQ ID NO:15 (H-CDR2) andSEQ ID NO:16 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:18 (L-CDR1), SEQ ID NO:19 (L-CDR2) and SEQ IDNO:20 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:106 (H-CDR1), SEQ ID NO:107 (H-CDR2) andSEQ ID NO:108 (H-CDR3) and comprises a light chain antigen-bindingregion that comprises SEQ ID NO:110 (L-COR1). SEQ ID NO:111 (L-CDR2) andSEQ ID NO:112 (L-CDR3).

In a preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a heavy chain antigen-bindingregion that comprises SEQ ID NO:92 (H-CDR1), SEQ ID NO:93 (H-CDR2) andSEQ ID NO:94 (H-CDR3) and comprises a light chain antigen-binding regionthat comprises SEQ ID NO:96 (L-CDR1), SEQ ID NO:97 (L-CDR2) and SEQ IDNO:98 (L-CDR3).

Antibodies differ in sequence, not only within their complementaritydetermining regions (CDRs), but also in the framework (FR). Thesesequence differences are encoded in the different V-genes. The humanantibody germline repertoire has been completely sequenced. There areabout 50 functional VH germline genes which can be grouped into sixsubfamilies according to sequence homology VH1, VH2, VH3, VH4, VH5 andVH6 (Tomlinson et al., 1992, J. Mol. Biol. 227, 776-798; Matsuda &Honjo, 1996, Advan. Immunol. 62, 1-29). About 40 functional VL kappagenes comprising seven subfamilies are known (Cox et al., 1994, Eur. J.Immunol. 24, 827-836; Barbie & Lefranc, 1998, Exp. Clin. Immunogenet.15, 171-183): Vkappa1, Vkappa2, Vkappa3, Vkappa4, Vkappa5, Vkappa6 andVkappa7. Disclosed herein are heavy chains of antibodies of thisinvention that belong to the human VH2 subfamily and the light chains ofantibodies of this invention that belong to the human Vkappal subfamily,respectively. It is known that framework sequences of antibodiesbelonging to the same subfamily are closely related, e.g. antibodiescomprising a human VH3 subfamily member all share comparable stability(Honegger et al., 2009, Protein Eng Des Sel. 22(3):121-134). It is wellknown in the art that CDRs from antibodies can be grafted on differentframeworks while maintaining special features of the correspondingorigin antibody. CDRs have been successfully grafted on frameworksbelonging to a different species as well as on frameworks of the samespecies belonging to a different subfamily. In a further embodiment theantibody or antigen-binding fragment of the invention comprises at leastone CDR sequence of antibody of the invention as depicted in Table 1 anda human variable chain framework sequence.

In a preferred embodiment the antibody or antigen-binding fragment ofthe invention comprises a variable light chain or light chainantigen-binding region comprising the L-CDR1, L-CDR2 and L-CDR3 sequenceof the variable light chain and a variable heavy chain or heavy chainantigen-binding region comprising the H-CDR1, H-CDR2 and H-CDR3 sequenceof the variable heavy chain antibody of the invention as depicted inTable 1 and a human variable light and human variable heavy chainframework sequence.

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:3 (VH) and a variable light chainsequences as presented by SEQ ID NO:7 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:33 (VH) and a variable light chainsequences as presented by SEQ ID NO:37 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:47 (VH) and a variable light chainsequences as presented by SEQ ID NO:51 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:119 (VH) and a variable light chainsequences as presented by SEQ ID NO:123 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:23 (VH) and a variable light chainsequences as presented by SEQ ID NO:27 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:75 (VH) and a variable light chainsequences as presented by SEQ ID NO:79 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:133 (VH) and a variable light chainsequences as presented by SEQ ID NO:137 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:147 (VH) and a variable light chainsequences as presented by SEQ ID NO:151 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:13 (VH) and a variable light chainsequences as presented by SEQ ID NO:17 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:61 (VH) and a variable light chainsequences as presented by SEQ ID NO:65 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:105 (VH) and a variable light chainsequences as presented by SEQ ID NO:109 (VL).

In a highly preferred embodiment the antibody of the invention orantigen-binding fragment thereof comprises a variable heavy chainsequence as presented by SEQ ID NO:91 (VH) and a variable light chainsequences as presented by SEQ ID NO:95 (VL).

In some embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereof binds toan epitope of human CEACAM6, wherein said epitope comprises one or moreamino acid residues selected from the group consisting of Gln60, Asn61,Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 ofSEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereof binds toan epitope of human CEACAM6, wherein said epitope comprises the aminoacid residues Gln60, Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90,Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In some embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereofinteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises one, two, three four, five, eight, ten, fifteen ormore amino acid residues selected from the group consisting of Pro59,Gln60, Asn61, Arg62, Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88,Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereofinteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises the amino acid residues Pro59, Gln60, Asn61, Arg62,Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125,Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In some embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereof binds toan epitope of human CEACAM6, wherein said epitope comprises one or moreamino acid residues selected from the group consisting of Gln60, Asn61,Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 ofSEQ ID NO: 179, and wherein said antibody or antigen-binding antibodyfragment thereof, or variant thereof binds to a human CEACAM6 proteincomprising an IIe63Leu mutation and does not bind to a human CEACAM6protein comprising an IIe63Phe mutation (numbering according toSEQ-ID:179).

In certain embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereof binds toan epitope of human CEACAM6, wherein said epitope comprises the aminoacid residues Gln60, Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90,Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein said antibodyor antigen-binding antibody fragment thereof, or variant thereof bindsto a human CEACAM6 protein comprising an IIe63Leu mutation and does notbind to a human CEACAM6 protein comprising an IIe63Phe mutation(numbering according to SEQ-ID:179).

In some embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereofinteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises one, two, three four, five, eight, ten, fifteen ormore amino acid residues selected from the group consisting of Pro59,Gln60, Asn61, Arg62, Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88,Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179, andwherein said antibody or antigen-binding antibody fragment thereof, orvariant thereof binds to a human CEACAM6 protein comprising an IIe63Leumutation and does not bind to a human CEACAM6 protein comprising anIIe63Phe mutation (numbering according to SEQ-ID:179).

In certain embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereofinteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises the amino acid residues Pro59, Gln60, Asn61, Arg62,Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125,Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein said antibodyor antigen-binding antibody fragment thereof, or variant thereof bindsto a human CEACAM6 protein comprising an Ile63Leu mutation and does notbind to a human CEACAM6 protein comprising an Ile63Phe mutation(numbering according to SEQ-ID:179).

In some embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereof binds toan epitope of human CEACAM6, wherein said epitope comprises one or moreamino acid residues selected from the group consisting of Gln60, Asn61,Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 ofSEQ ID NO: 179, and wherein said antibody or antigen-binding antibodyfragment thereof, or variant thereof competes for binding to CECEAM6with an antibody comprising a variable heavy chain sequence as presentedby SEQ ID NO:47 (VH) and a variable light chain sequences as presentedby SEQ ID NO:51 (VL).

In certain embodiments, the anti-CEACAM6 antibody of the invention bindsto an epitope of human CEACAM6, wherein said epitope comprises the aminoacid residues Gln60, Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90,Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein said antibodyor antigen-binding antibody fragment thereof, or variant thereofcompetes for binding to CECEAM6 with an antibody comprising a variableheavy chain sequence as presented by SEQ ID NO:47 (VH) and a variablelight chain sequences as presented by SEQ ID NO:51 (VL).

In some embodiments, the anti-CEACAM6 antibody of the inventioninteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises one, two, three four, five, eight, ten, fifteen ormore amino acid residues selected from the group consisting of Pro59,Gln60, Asn61, Arg62, Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88,Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179, andwherein said antibody or antigen-binding antibody fragment thereof, orvariant thereof competes for binding to CECEAM6 with an antibodycomprising a variable heavy chain sequence as presented by SEQ ID NO:47(VH) and a variable light chain sequences as presented by SEQ ID NO:51(VL).

In certain embodiments, the anti-CEACAM6 antibody of the inventioninteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises the amino acid residues Pro59, Gln60, Asn61, Arg62,Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125,Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein said antibodyor antigen-binding antibody fragment thereof, or variant thereofcompetes for binding to CECEAM6 with an antibody comprising a variableheavy chain sequence as presented by SEQ ID NO:47 (VH) and a variablelight chain sequences as presented by SEQ ID NO:51 (VL).

In some embodiments, the anti-CEACAM6 antibody of the invention or anantigen-binding antibody fragment thereof, or a variant thereof binds toan epitope of human CEACAM6, wherein said epitope comprises one or moreamino acid residues selected from the group consisting of Gln60, Asn61,Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 ofSEQ ID NO: 179, and wherein said antibody or antigen-binding antibodyfragment thereof, or variant thereof competes for binding to CECEAM6with an antibody comprising a variable heavy chain sequence as presentedby SEQ ID NO:47 (VH) and a variable light chain sequences as presentedby SEQ ID NO:51 (VL), and wherein said antibody or antigen-bindingantibody fragment thereof, or variant thereof binds to a human CEACAM6protein comprising an Ile63Leu mutation and does not bind to a humanCEACAM6 protein comprising an Ile63Phe mutation (numbering according toSEQ-ID:179).

In certain embodiments, the anti-CEACAM6 antibody of the invention bindsto an epitope of human CEACAM6, wherein said epitope comprises the aminoacid residues Gln60, Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90,Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein said antibodyor antigen-binding antibody fragment thereof, or variant thereofcompetes for binding to CECEAM6 with an antibody comprising a variableheavy chain sequence as presented by SEQ ID NO:47 (VH) and a variablelight chain sequences as presented by SEQ ID NO:51 (VL) and wherein saidantibody or antigen-binding antibody fragment thereof, or variantthereof binds to a human CEACAM6 protein comprising an Ile63Leu mutationand does not bind to a human CEACAM6 protein comprising an Ile63Phemutation (numbering according to SEQ-ID:179).

In some embodiments, the anti-CEACAM6 antibody of the inventioninteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises one, two, three four, five, eight, ten, fifteen ormore amino acid residues selected from the group consisting of Pro59,Gln60, Asn61, Arg62, Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88,Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179, andwherein said antibody or antigen-binding antibody fragment thereof, orvariant thereof competes for binding to CECEAM6 with an antibodycomprising a variable heavy chain sequence as presented by SEQ ID NO:47(VH) and a variable light chain sequences as presented by SEQ ID NO:51(VL) and wherein said antibody or antigen-binding antibody fragmentthereof, or variant thereof binds to a human CEACAM6 protein comprisingan Ile63Leu mutation and does not bind to a human CEACAM6 proteincomprising an Ile63Phe mutation (numbering according to SEQ-ID:179).

In certain embodiments, the anti-CEACAM6 antibody of the inventioninteracts with, e.g. binds to, an epitope of human CEACAM6, wherein saidepitope comprises the amino acid residues Pro59, Gln60, Asn61, Arg62,Ile63, Gly64, Va183, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125,Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein said antibodyor antigen-binding antibody fragment thereof, or variant thereofcompetes for binding to CECEAM6 with an antibody comprising a variableheavy chain sequence as presented by SEQ ID NO:47 (VH) and a variablelight chain sequences as presented by SEQ ID NO:51 (VL) and wherein saidantibody or antigen-binding antibody fragment thereof, or variantthereof binds to a human CEACAM6 protein comprising an Ile63Leu mutationand does not bind to a human CEACAM6 protein comprising an Ile63Phemutation (numbering according to SEQ-ID:179).

An antibody of the invention may be an IgG (immunoglobulin G e.g. IgG1IgG2, IgG3, IgG4) or IgA, IgD, IgE, IgM, while an antibody fragment maybe a Fab, Fab′, F(ab′)₂, Fab′-SH or scFv, for example. An inventiveantibody fragment, accordingly, may be, or may contain, anantigen-binding region that behaves in one or more ways as describedherein.

In a preferred embodiment the antibodies or antigen-binding antibodyfragments of the invention are monoclonal.

In some embodiments antibodies of the invention or antigen-bindingfragments thereof, or nucleic acids encoding the same are isolated. Anisolated biological component (such as a nucleic acid molecule orprotein such as an antibody) is one that has been substantiallyseparated or purified away from other biological components in the cellof the organism in which the component naturally occurs, e.g., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Antibody Generation

An antibody of the invention may be derived from a recombinant antibodylibrary that is based on amino acid sequences that have been isolatedfrom the antibodies of a large number of healthy volunteers e.g. usingthe n-CoDeR® technology the fully human CDRs are recombined into newantibody molecules (Carlson & Soderlind, Expert Rev Mol Diagn. 2001 May;1(1):102-8). Or alternatively for example antibody libraries as thefully human antibody phage display library described in Hoet R M et al.,Nat Biotechnol 2005; 23(3):344-8) can be used to isolateCEACAM6-specific antibodies. Antibodies or antibody fragments isolatedfrom human antibody libraries are considered human antibodies or humanantibody fragments herein.

Human antibodies may be further prepared by administering an immunogento a transgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. For exampleimmunization of genetically engineered mice inter alia immunization ofhMAb mice (e.g. Veloclmmune mouse® or XENOMOUSE®) may be performed.

Further antibodies may be generated using the hybridoma technology (forexample see Kohler and Milstein Nature. 1975 Aug. 7 256(5517):495-7),resulting in for example murine, rat, or rabbit antibodies which can beconverted into chimeric or humanized antibodies. Humanized antibodiesand methods of making them are reviewed, e.g., in Almagro and Fransson,Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., inRiechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. NatlAcad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34(2005) (describing specificity determining region (SDR) grafting);Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”);Dall' Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”);and Osboum et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.Cancer, 83:252-260 (2000) (describing the “guided selection” approach toFR shuffling).

Examples are provided for the generation of antibodies using arecombinant antibody library and immunization of mice combined withsubsequent humanization.

It is a further aspect of the invention to provide a method to generateantibodies specifically binding to human CEACAM6 and to Macacafascicularis CEACAM6, which do not significantly cross-react with humanCEACAM1, human CEACAM3, and human CEACAM5. It is an embodiment of theinvention to provide a method for generation of anti-CEACAM6 antibodiescharacterized by comprising the steps of immunization of an animal,preferentially a mouse, with cynomolgus CECAM6 domain 1 (represented byamino acids 35-142 of SEQ-ID NO:177), determining the amino acidsequence of antibodies specifically binding to human CEACAM6 and tocynomolgus CEACAM6, followed optionally by humanization or generation ofa chimeric antibody, and expression of said antibodies. The expressionsystem can be a recombinant or a cell free expression system. Suitablehost cells for recombinant expression are prokaryotic and eukaryoticcells. Preferred are mammalian expression systems.

Peptide Variants

Antibodies or antigen-binding fragments of the invention are not limitedto the specific peptide sequences provided herein. Rather, the inventionalso embodies variants of these polypeptides. With reference to theinstant disclosure and conventionally available technologies andreferences, the skilled worker will be able to prepare, test and utilizefunctional variants of the antibodies disclosed herein, whileappreciating these variants having the ability to bind to CEACAM6 fallwithin the scope of the present invention.

A variant can include, for example, an antibody that has at least onealtered complementary determining region (CDR) (hyper-variable) and/orframework (FR) (variable) domain/position, vis-a-vis a peptide sequencedisclosed herein.

By altering one or more amino acid residues in a CDR or FR region, theskilled worker routinely can generate mutated or diversified antibodysequences, which can be screened against the antigen, for new orimproved properties, for example.

A further preferred embodiment of the invention is an antibody orantigen-binding fragment in which the VH and VL sequences are selectedas shown in Table 1. The skilled worker can use the data in Table 1 todesign peptide variants that are within the scope of the presentinvention. It is preferred that variants are constructed by changingamino acids within one or more CDR regions; a variant might also haveone or more altered framework regions. Alterations also may be made inthe framework regions. For example, a peptide FR domain might be alteredwhere there is a deviation in a residue compared to a germline sequence.

Alternatively, the skilled worker could make the same analysis bycomparing the amino acid sequences disclosed herein to known sequencesof the same class of such antibodies, using, for example, the proceduredescribed by Knappik A., et al., JMB 2000, 296:57-86.

Furthermore, variants may be obtained by using one antibody as startingpoint for further optimization by diversifying one or more amino acidresidues in the antibody, preferably amino acid residues in one or moreCDRs, and by screening the resulting collection of antibody variants forvariants with improved properties. Particularly preferred isdiversification of one or more amino acid residues in CDR3 of VL and/orVH. Diversification can be done e.g. by synthesizing a collection of DNAmolecules using trinucleotide mutagenesis (TRIM) technology (Virnekäs B.et al., Nucl. Acids Res. 1994, 22: 5600.). Antibodies or antigen-bindingfragments thereof include molecules with modifications/variationsincluding but not limited to e.g. modifications leading to alteredhalf-life (e.g. modification of the Fc part or attachment of furthermolecules such as PEG), altered binding affinity or altered ADCC or CDCactivity.

Conservative Amino Acid Variants

Polypeptide variants may be made that conserve the overall molecularstructure of an antibody peptide sequence described herein. Given theproperties of the individual amino acids, some rational substitutionswill be recognized by the skilled worker. Amino acid substitutions,i.e., “conservative substitutions,” may be made, for instance, on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophane, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found inα-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in β-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants.

Glycosylation Variants

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 using Kabat EU numbering ofthe CH2 domain of the Fc region; see, e.g., Wright et al. TrendsBiotechnol. 15: 26-32 (1997).

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the expression system (e.g. hostcell) and/or by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

In one embodiment of this invention, aglycosyl antibodies havingdecreased effector function or antibody derivatives are prepared byexpression in a prokaryotic host. Suitable prokaryotic hosts for includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

In one embodiment, antibody variants are provided having decreasedeffector function, which are characterized by a modification at theconserved N-linked site in the CH2 domains of the Fc portion of saidantibody. In one embodiment of present invention, the modificationcomprises a mutation at the heavy chain glycosylation site to preventglycosylation at the site. Thus, in one preferred embodiment of thisinvention, the aglycosyl antibodies or antibody derivatives are preparedby mutation of the heavy chain glycosylation site, - i.e., mutation ofN297 using Kabat EU numbering and expressed in an appropriate host cell.

In another embodiment of the present invention, aglycosyl antibodies orantibody derivatives have decreased effector function, wherein themodification at the conserved N-linked site in the CH2 domains of the Fcportion of said antibody or antibody derivative comprises the removal ofthe CH2 domain glycans, - i.e., deglycosylation. These aglycosylantibodies may be generated by conventional methods and then deglycosylated enzymatically. Methods for enzymatic deglycosylation ofantibodies are well known in the art (e.g. Winkelhake & Nicolson (1976),J Biol Chem. 251(4):1074-80).

In another embodiment of this invention, deglycosylation may be achievedusing the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983),Proc Natl Acad Sci USA, 80(21):6632-6). That is, the modification is theprevention of glycosylation at the conserved N-linked site in the CH2domains of the Fc portion of said antibody.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.

Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: Okazaki et al. J Mol.Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

Examples of cell lines capable of producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); and WO 2004/056312), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006)).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO1997/30087; WO1998/58964; and WO1999/22764.

Fc Region Variants

In certain embodiments, one or more amino acid modifications (e.g. asubstitution) may be introduced into the Fc region of an antibody (e.g.,a human IgG1, IgG2, IgG3 or IgG4 Fc region) provided herein, therebygenerating an Fc region variant.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. In some embodiments, alterations are madein the Fc region that result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC).

In certain embodiments, the invention contemplates an antibody variantthat possesses an increased or decreased half-live. Antibodies withincreased half-lives and improved binding to the neonatal Fc receptor(FcRn), which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al., JImmunol. 24:249 (1994)), are described in US2005/0014934 (Hinton etal.). Those antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.

Antibody-Drug Conjugates (ADC)

The invention also provides antibody-drug conjugates (ADC,immunoconjugates) comprising an anti-CEACAM6 antibody conjugated to oneor more cytotoxic agents, such as chemotherapeutic agents or drugs,growth inhibitory agents, toxins (e.g., protein toxins, enzymaticallyactive toxins of bacterial, fungal, plant, human or animal origin, orfragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP0425235); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof; an anthracycline such as daunomycinor doxorubicin; methotrexate; vindesine; a taxane such as docetaxel,paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; andCC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alphasarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (P API, P APII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include ²²⁷Th, ²²⁵Ac, ²¹¹At,¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Si, ³²P, ²¹²Pb and radioactiveisotopes of Lu. When the radioconjugate is used for detection, it maycomprise a radioactive atom for scintigraphic studies, for exampleTc99m, or a spin label for nuclear magnetic resonance (NMR) imaging,such as iodine-123 again, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

The linker may be a “cleavable linker” facilitating release of acytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52: 12 7-131(1992).

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

DNA Molecules of the Invention

The present invention also relates to the DNA molecules that encode anantibody of the invention or antigen-binding fragment thereof. The DNAsequences used for the antibodies expressed are given in Table 32. Thesesequences are optimized in certain cases for mammalian expression. DNAmolecules of the invention are not limited to the sequences disclosedherein, but also include variants thereof. DNA variants within theinvention may be described by reference to their physical properties inhybridization. The skilled worker will recognize that DNA can be used toidentify its complement and, since DNA is double stranded, itsequivalent or homolog, using nucleic acid hybridization techniques. Italso will be recognized that hybridization can occur with less than 100%complementarity. However, given appropriate choice of conditions,hybridization techniques can be used to differentiate among DNAsequences based on their structural relatedness to a particular probe.For guidance regarding such conditions see, Sambrook et al., 1989 supraand Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995).Current Protocols in Molecular Biology. New York: John Wiley and Sons).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

Functionally Equivalent DNA Variants

Yet another class of DNA variants within the scope of the invention maybe described with reference to the product they encode. Thesefunctionally equivalent polynucleotides are characterized by the factthat they encode the same peptide sequences due to the degeneracy of thegenetic code.

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides are widely available. See Ausubel et al.,section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may besynthesized and assembled in a fashion first reported by Khorana et al.,J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section8.2. Synthetic DNAs preferably are designed with convenient restrictionsites engineered at the 5′ and 3′ ends of the gene to facilitate cloninginto an appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typicalmethod, a target DNA is cloned into a single-stranded DNA bacteriophagevehicle. Single-stranded DNA is isolated and hybridized with anoligonucleotide containing the desired nucleotide alteration(s). Thecomplementary strand is synthesized and the double stranded phage isintroduced into a host. Some of the resulting progeny will contain thedesired mutant, which can be confirmed using DNA sequencing. Inaddition, various methods are available that increase the probabilitythat the progeny phage will be the desired mutant. These methods arewell known to those in the field and kits are commercially available forgenerating such mutants.

Recombinant DNA Constructs and Expression

The present invention further provides recombinant DNA constructscomprising one or more of the nucleotide sequences of the presentinvention (see Table 32). The recombinant constructs of the presentinvention can be used in connection with a vector, such as a plasmid,phagemid, phage or viral vector, into which a DNA molecule encoding anantibody of the invention or antigen-binding fragment thereof or variantthereof is inserted.

An antibody, antigen binding portion, or variant thereof provided hereincan be prepared by recombinant expression of nucleic acid sequencesencoding light and heavy chains or portions thereof in a host cell. Toexpress an antibody, antigen binding portion, or variant thereofrecombinantly a host cell can be transfected with one or morerecombinant expression vectors carrying DNA fragments encoding the lightand/or heavy chains or portions thereof such that the light and heavychains are expressed in the host cell. Standard recombinant DNAmethodologies are used to prepare and/or obtain nucleic acids encodingthe heavy and light chains, incorporate these nucleic acids intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds.),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.No. 4,816,397 by Boss et al..

In addition, the nucleic acid sequences encoding variable regions of theheavy and/or light chains can be converted, for example, to nucleic acidsequences encoding full-length antibody chains, Fab fragments, or toscFv. The VL- or VH-encoding DNA fragment can be operatively linked,(such that the amino acid sequences encoded by the two DNA fragments arein-frame) to another DNA fragment encoding, for example, an antibodyconstant region or a flexible linker. The sequences of human heavy chainand light chain constant regions are known in the art (see e.g., Kabat,E. A., el al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification.

To create a polynucleotide sequence that encodes a scFv, the VH- andVL-encoding nucleic acids can be operatively linked to another fragmentencoding a flexible linker such that the VH and VL sequences can beexpressed as a contiguous single-chain protein, with the VL and VHregions joined by the flexible linker (see e.g., Bird et al. (1988)Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, antigen binding fragments thereof or variantsthereof standard recombinant DNA expression methods can be used (see,for example, Goeddel; Gene Expression Technology. Methods in Enzymology185, Academic Press, San Diego, Calif. (1990)). For example, DNAencoding the desired polypeptide can be inserted into an expressionvector which is then transfected into a suitable host cell. Suitablehost cells are prokaryotic and eukaryotic cells. Examples forprokaryotic host cells are e.g. bacteria, examples for eukaryotic hostscells are yeasts, insects and insect cells, plants and plant cells,transgenic animals, or mammalian cells. In some embodiments, the DNAsencoding the heavy and light chains are inserted into separate vectors.In other embodiments, the DNA encoding the heavy and light chains isinserted into the same vector. It is understood that the design of theexpression vector, including the selection of regulatory sequences isaffected by factors such as the choice of the host cell, the level ofexpression of protein desired and whether expression is constitutive orinducible.

Therefore, an embodiment of the present invention are also host cellscomprising the vector or a nucleic acid molecule, whereby the host cellcan be a higher eukaryotic host cell, such as a mammalian cell, a lowereukaryotic host cell, such as a yeast cell, and may be a prokaryoticcell, such as a bacterial cell.

Another embodiment of the present invention is a method of using thehost cell to produce an antibody and antigen binding fragments,comprising culturing the host cell under suitable conditions andrecovering said antibody.

Therefore another embodiment of the present invention is the productionof the antibodies according to this invention with the host cells of thepresent invention and purification of these antibodies to at least 95%homogeneity by weight.

Bacterial Expression

Useful expression vectors for bacterial use are constructed by insertinga DNA sequence encoding a desired protein together with suitabletranslation initiation and termination signals in operable reading phasewith a functional promoter. The vector will comprise one or morephenotypic selectable markers and an origin of replication to ensuremaintenance of the vector and, if desirable, to provide amplificationwithin the host. Suitable prokaryotic hosts for transformation includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker and abacterial origin of replication derived from commercially availableplasmids typically containing elements of the well-known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Therefore, an embodiment of the present invention is an expressionvector comprising a nucleic acid sequence encoding for the novelantibodies of the present invention.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic host, including, for example, E. coli,Bacillus subtilis, Salmonella typhimurium and various species within thegenera Pseudomonas, Streptomyces, and Staphylococcus, preferably, fromE. coli cells.

Mammalian Expression

Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Expression of theantibodies may be constitutive or regulated (e.g. inducible by additionor removal of small molecule inductors such as Tetracyclin inconjunction with Tet system). For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al.. The recombinant expressionvectors can also include origins of replication and selectable markers(see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and U.S. Pat. No.5,179,017). Suitable selectable markers include genes that conferresistance to drugs such as G418, puromycin, hygromycin, blasticidin,zeocin/bleomycin or methotrexate or selectable marker that exploitauxotrophies such as Glutamine Synthetase (Bebbington et al.,Biotechnology (N Y). 1992 Feb;10(2):169-75), on a host cell into whichthe vector has been introduced. For example, the dihydrofolate reductase(DHFR) gene confers resistance to methotrexate, neo gene confersresistance to G418, the bsd gene from Aspergillus terreus confersresistance to blasticidin, puromycin N-acetyl-transferase confersresistance to puromycin, the Sh ble gene product confers resitance tozeocin, and resistance to hygromycin is conferred by the E. colihygromycin resistance gene (hyg or hph). Selectable markers like DHFR orGlutamine Synthetase are also useful for amplification techniques inconjunction with MTX and MSX.

Transfection of the expression vector into a host cell can be carriedout using standard techniques such as electroporation, nucleofection,calcium-phosphate precipitation, lipofection, polycation-basedtransfection such as polyethlylenimine (PEI)-based transfection andDEAE-dextran transfection.

Suitable mammalian host cells for expressing the antibodies, antigenbinding fragments thereof or variants thereof provided herein includeChinese Hamster Ovary (CHO cells) such as CHO-K1, CHO-S, CHO-K1SV[including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 June;33(2):405-12, used with a DHFR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621; and otherknockout cells exemplified in Fan et al., Biotechnol Bioeng. 2012 April;109(4):1007-15], NSO myeloma cells, COS cells, HEK293 cells, HKB11cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

Expression might also be transient or semi-stable in expression systemssuch as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E,HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S,CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (forinstance Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9).

In some embodiments, the expression vector is designed such that theexpressed protein is secreted into the culture medium in which the hostcells are grown. The antibodies, antigen binding fragments thereof orvariants thereof can be recovered from the culture medium using standardprotein purification methods.

Purification

Antibodies of the invention or antigen-binding fragments thereof orvariants thereof can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to ammoniumsulfate or ethanol precipitation, acid extraction, Protein Achromatography, Protein G chromatography, anion or cation exchangechromatography, phospho-cellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. High performance liquidchromatography (“HPLC”) can also be employed for purification. See,e.g., Colligan, Current Protocols in Immunology, or Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g.,Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein byreference.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from an eukaryotic host, including, for example, yeast,higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated. Suchmethods are described in many standard laboratory manuals, such asSambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12,13, 16, 18 and 20.

In preferred embodiments, the antibody is purified (1) to greater than95% by weight of antibody as determined e.g. by the Lowry method, UV-Visspectroscopy or by by SDS-Capillary Gel electrophoresis (for example ona Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and infurther preferred embodiments more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated naturally occurring antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount of an antibody or anantigen-binding fragment thereof or a variant thereof contemplated bythe invention. A “therapeutically effective” amount hereby is defined asthe amount of an antibody or antigen-binding fragment that is ofsufficient quantity to reduce proliferation of CEACAM6 positive cell orto reduce size of a CEACAM6 expressing tumor in a treated area of asubject - either as a single dose or according to a multiple doseregimen, alone or in combination with other agents, which leads to thealleviation of an adverse condition, yet which amount is toxicologicallytolerable. The subject may be a human or non-human animal (e.g., rabbit,rat, mouse, dog, monkey or other lower-order primate).

It is an embodiment of the invention to provide an antibody orantigen-binding fragment thereof for use as medicament.

It is an embodiment of the invention to provide an antibody orantigen-binding fragment thereof for use as a medicament for thetreatment of cancer. In a preferred embodiment the cancer is a tumor andin a highly preferred embodiment the cancer is a solid tumor.

It is an embodiment of the invention to use the antibody orantigen-binding fragment thereof in the manufacture of a medicament forthe treatment of a disease.

It is an embodiment of the invention to use the antibody orantigen-binding fragment thereof in the manufacture of a medicament forthe treatment of cancer. In a preferred embodiment the cancer is a tumorand in a highly preferred embodiment the cancer is a solid tumor.

The inventive antibodies or antigen-binding fragments thereof can beused as a therapeutic or a diagnostic tool in a variety of situationswith aberrant CEACAM6-signaling, e.g. cell proliferative disorders suchas cancer or fibrotic diseases. Disorders and conditions particularlysuitable for treatment with an antibody of the inventions are solidtumors, such as cancers of the breast, respiratory tract, brain,reproductive organs, digestive tract, urinary tract, eye, liver, skin,head and neck, thyroid, parathyroid, and their distant metastases. Thosedisorders also include lymphomas, sarcomas and leukemias.

Tumors of the digestive tract include, but are not limited to anal,colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal,small-intestine, and salivary gland cancers.

Examples of esophageal cancer include, but are not limited to esophagealcell carcinomas and adenocarcinomas, as well as squamous cellcarcinomas, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma andlymphoma,.

Examples of gastric cancer include, but are not limited to intestinaltype and diffuse type gastric adenocarcinoma.

Examples of pancreatic cancer include, but are not limited to ductaladenocarcinoma, adenosquamous carcinomas and pancreatic endocrinetumors.

Examples of breast cancer include, but are not limited to triplenegative breast cancer, invasive ductal carcinoma, invasive lobularcarcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are notlimited to small-cell and non-small-cell lung carcinoma, as well asbronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to brain stem andhypophtalmic glioma, cerebellar and cerebral astrocytoma, glioblastoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumor.

Tumors of the male reproductive organs include, but are not limited toprostate and testicular cancer. Tumors of the female reproductive organsinclude, but are not limited to endometrial, cervical, ovarian, vaginaland vulvar cancer, as well as sarcoma of the uterus.

Examples of ovarian cancer include, but are not limited to seroustumour, endometrioid tumor, mucinous cystadenocarcinoma, granulosa celltumor, Sertoli-Leydig cell tumor and arrhenoblastoma

Examples of cervical cancer include, but are not limited to squamouscell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cellcarcinoma, neuroendocrine tumour, glassy cell carcinoma andvilloglandular adenocarcinoma.

Tumors of the urinary tract include, but are not limited to bladder,penile, kidney, renal pelvis, ureter, urethral, and hereditary andsporadic papillary renal cancers.

Examples of kidney cancer include, but are not limited to renal cellcarcinoma, urothelial cell carcinoma, juxtaglomerular cell tumor(reninoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma,clear-cell sarcoma of the kidney, mesoblastic nephroma and Wilms' tumor.

Examples of bladder cancer include, but are not limited to transitionalcell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma andsmall cell carcinoma.

Eye cancers include, but are not limited to intraocular melanoma andretinoblastoma.

Examples of liver cancers include, but are not limited to hepatocellularcarcinoma (liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to squamous cellcancer of the head and neck, laryngeal, hypopharyngeal, nasopharyngeal,oropharyngeal cancer, salivary gland cancer, lip and oral cavity cancer,and squamous cell cancer.

Lymphomas include, but are not limited to AIDS-related lymphoma,non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma,Hodgkin's disease, and lymphoma of the central nervous system.

Sarcomas include, but are not limited to sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma.

Leukemias include, but are not limited to acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

In a preferred embodiment, the antibodies of the invention orantigen-binding fragments thereof are suitable for a therapeutic ordiagnostic method for the treatment or diagnosis of a cancer diseasecomprised in a group consisting of colorectal cancer, non-small-celllung cancer (NSCLC), small cell lung cancer (SCLC), pancreatic cancer,gastric cancer, breast cancer and multiple myeloma.

In addition, the inventive antibodies or antigen-binding fragmentsthereof can also be used as a therapeutic or a diagnostic tool in avariety of other disorders wherein CEACAM6 is involved such as, but notlimited to lung infection e.g. influenza,

Crohns disease, inflammatory bowel disease, psoriasis, lung cysticfibrosis, prevention of bacterial docking to GI-intract, trauma,bleeding burn, surgery, stroke, myocardial infarction, sepsis,pneumonia, vaccination for infection & cancer, chronic virus infection.

The disorders mentioned above have been well characterized in humans,but also exist with a similar etiology in other animals, includingmammals, and can be treated by administering pharmaceutical compositionsof the present invention.

An antibody of the invention or an antigen-binding fragment thereof or avariant thereof might be co-administered with known medicaments, and insome instances the antibody might itself be modified. For example, anantibody or an antigen-binding fragment thereof or a variant thereofcould be conjugated to a cytotoxic agent or radioisotope to potentiallyfurther increase efficacy.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof may be administered as the sole pharmaceutical agentor in combination with one or more additional therapeutic agents wherethe combination causes no unacceptable adverse effects. This combinationtherapy includes administration of a single pharmaceutical dosageformulation which contains an antibody of the invention or anantigen-binding fragment thereof or a variants thereof and one or moreadditional therapeutic agents, as well as administration of an antibodyof the invention and each additional therapeutic agent in its ownseparate pharmaceutical dosage formulation. For example, an antibody ofthe invention or an antigen-binding fragment thereof or a variantthereof and a therapeutic agent may be administered to the patienttogether in a single liquid composition, or each agent may beadministered in separate dosage formulation.

Where separate dosage formulations are used, an antibody of theinvention or an antigen-binding fragment thereof or a variants thereofand one or more additional therapeutic agents may be administered atessentially the same time (e.g., concurrently) or at separatelystaggered times (e.g., sequentially).

In particular, antibodies of the present invention or antigen-bindingfragments thereof or variants thereof may be used in fixed or separatecombination with other anti-tumor agents such as alkylating agents,anti-metabolites, plant-derived anti-tumor agents, hormonal therapyagents, topoisomerase inhibitors, immunologicals, antibodies, antibodydrugs, biological response modifiers, anti-angiogenic compounds, celltherapies, and other anti-tumor drugs including but not limited tocamptothecin derivatives, kinase inhibitors, targeted drugs.

In this regard, the following is a non-limiting list of examples ofsecondary agents that may be used in combination with the antibodies ofthe present invention:

Alkylating agents include, but are not limited to, nitrogen mustardN-oxide, cyclophosphamide, ifosfamide, thiotepa, ranimustine, nimustine,temozolomide, altretamine, apaziquone, brostallicin, bendamustine,carmustine, estramustine, fotemustine, glufosfamide, mafosfamide,bendamustin, and mitolactol; platinum-coordinated alkylating compoundsinclude, but are not limited to, cisplatin, carboplatin, eptaplatin,lobaplatin, nedaplatin, oxaliplatin, and satraplatin;

Anti-metabolites include, but are not limited to, methotrexate,6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil alone or incombination with leucovorin, tegafur, doxifluridine, carmofur,cytarabine, cytarabine ocfosfate, enocitabine, gemcitabine, fludarabin,5-azacitidine, capecitabine, cladribine, clofarabine, decitabine,eflornithine, ethynylcytidine, cytosine arabinoside, hydroxyurea,melphalan, nelarabine, nolatrexed, ocfosfite, disodium premetrexed,pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate,vidarabine, vincristine, and vinorelbine;

Hormonal therapy agents include, but are not limited to, exemestane,Lupron, anastrozole, doxercalciferol, fadrozole, formestane, 11-betahydroxysteroid dehydrogenase 1 inhibitors, 17-alpha hydroxylase/17,20lyase inhibitors such as abiraterone acetate, 5-alpha reductaseinhibitors such as finasteride and epristeride, anti-estrogens such astamoxifen citrate and fulvestrant, Trelstar, toremifene, raloxifene,lasofoxifene, letrozole, anti-androgens such as bicalutamide, flutamide,mifepristone, nilutamide, Casodex, and anti-progesterones andcombinations thereof;

Plant-derived anti-tumor substances include, e.g., those selected frommitotic inhibitors, for example epothilones such as sagopilone,ixabepilone and epothilone B, vinblastine, vinflunine, docetaxel, andPaclitaxel;

Cytotoxic topoisomerase inhibiting agents include, but are not limitedto, aclarubicin, doxorubicin, amonafide, belotecan, camptothecin,10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, Irinotecan,topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan,lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane,tafluposide, and combinations thereof;

Immunologicals include interferons such as interferon alpha, interferonalpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a andinterferon gamma-n1, GM-CSF and other immune enhancing agents such asL19-IL2 and other IL2 derivatives, filgrastim, lentinan, sizofilan,TheraCys, ubenimex, aldesleukin, alemtuzumab, BAM-002, dacarbazine,daclizumab, denileukin, gemtuzumab, ozogamicin, ibritumomab, imiquimod,lenograstim, lentinan, melanoma vaccine (Corixa), molgramostim,sargramostim, tasonermin, tecleukin, thymalasin, tositumomab, Vimlizin,epratuzumab, mitumomab, oregovomab, pemtumomab, and Provenge; ALNX6000,Urelumab, PF-005082566, Galunisertib, AZ10606120, NF340, BMS-777607;

Immunologicals also include drugs directed towards immune checkpointmodulators or co-inhibitory receptors including but not limited toCTLA-4, PD1, PD-L1, B7-H3 receptor, B7-H4 receptor, BTLA, TIM3, LAG3,KIRDL, 2B4, VISTA, CD244, CD160, TIGlT, CEACAM1, CEACAM5, HHLA2.Specifically some of these drugs are Ipilimumab, Tremelimumab,Nivolumab, Pembrolizumab, Pidilizumab, AMP-224, AMP-514, PDR001,MDX1105, BMS-936,559, Atezolizumab, Medi4736, Avelumab, MSB0010718C,MGA271, IMP321, BMS-986,016, Bavituximab, MNRP1685A, Celecoxib,PF-04418948, RQ-15986;

Immunologicals also include activators of co-stimulatory receptorsincluding drugs directed towards but not limited to CD28, ICOS, 4-1BB,0X40, CD27, KIRDS, GlTR, HVEM, TNFRSF25, CD4OL. TMIGD2, TIM-1, CEACAM1,CEACAM5. Among those drugs are CP-870893, Lucatumumab, Dacetuzumab,Anti-OX40, MEDI0562, MEDI6469, MEDI6383, CDX-1127, TRX518, Varlilumab;

Immunologicals also include agents that modulate Treg activity includingthose directed but not limited to FOXP3, CD25, CCR4. Among those agentsis daclizumab;

Immunologicals also include agents that modulate the activity of myeloidderived suppressor cells including those directed but not limited toCSF1R. An example is emactuzumab, Taladafil;

Immunologicals also include agents which modulate the innate immune cellresponse including agents directed to Toll-like receptors including butnot limited to TLR3, TLR4, TLR7, TLR8, TLR9, NGK2A, NKG2D. These drugsare for example Imiquimod, CPG7909 (PF-3512676, CPG2006); MGN1703,SD-101, hiltonol (Poly ICLC), Anti-NGK2A (IPH2201), OM-174, 852A,VTX-2337, IMO-2055;

Immunologicals also include drugs which modulate the innate immune cellresponse including drugs directed but not limited to CSF-1, CSF1R, KIR,ILTs, LIRs, MICA, MICB, CD244, CCL2, CD47. Specifically some of thesedrugs are Anti-KIR (IHP2101; IPH2101; 1-7F9); Lirilumab (IPH2012;BMS-986,015); Carlumab (CNT0888), IMC-CS4, FPA008, PLX3397, ARRY-382,CC-90002, Anti-CD47 (Hu5F9-G4), BLZ945;

Immunologicals also include agents for immune cell retargeting includingbut not limited to bispecific antibodies, Darts e.g. against B7-H3,Bites e.g CD19xCD3; e.g. Removab anti-EPCAMxCD3xFC, NK cell targetingagents;

Immunologicals also include agents which modulate the tumormicroenvironment and improve immune cell infiltration and responseincluding Vaccines and Adjuvants and which are not limited to GVAX,FVAX;

In this regard Vaccines comprise dendritic cell-based vaccines, viralvaccines, mRNA based vaxxines, multipeptide based vaccines;

Immunologicals also include agents for improved immune cell infiltrationincluding but not limited to IFN-g, IL15, IL21, IL2, CXCR4, CXC112, Someof these drugs are Denenicokin (BMS982, 470), ALT-803, hetIL15,Ulocuplumab, BKT140, CXCR2-specific mab, AD3100, Maravirox, PF-4136309;

Immunologicals also include agents or modalities which improve thepriming and activation of APCs and T cells including drugs directed tobut not limited to m-TOR GSK3beta inhibitors, loaded DCs (e.g.Provenge), Radiation therapy, external beam radiation;

Immunologicals also include Kynurenine pathway modulators includingdrugs directed to but not limited to ID01, ID02, TDO. Among those drugsare INCB024360, Indoximod (NLG8189; 1-methyl-D-tryptophan, D-1MT),GDC-0919, NLG919, LM10;

Immunologicals also include Adenosine Pathway modulators including drugsdirected to but not limited to CD39, CD73, A2A receptor, A2B receptor.Such drugs include Compound9, NCX-4016, AT38, SCH58261, SCH420814,PSB1115, ARL67176, AMPCP;

Immunologicals also include TGFbeta/ALKS pathway modulator. Such drugsinclude OY2157299, EW-7187;

Immunologicals also include Sting activators;

Biological response modifiers are agents that modify defense mechanismsof living organisms or biological responses such as survival, growth ordifferentiation of tissue cells to direct them to have anti-tumoractivity; such agents include, e.g., krestin, lentinan, sizofiran,picibanil, ProMune, and ubenimex;

Anti-angiogenic compounds include, but are not limited to, acitretin,aflibercept, angiostatin, aplidine, asentar, axitinib, bevacizumab,brivanib alaninat, cilengtide, combretastatin, endostatin, fenretinide,halofuginone, pazopanib, ranibizumab, rebimastat, recentin, regorafenib,removab, revlimid, sorafenib, squalamine, sunitinib, telatinib,thalidomide, ukrain, vatalanib, and vitaxin;

Antiangiogenic agents also include VEGF inhibitors including but notlimited to sorafenib, regorafenib, bevacizumab, sunitinib, recentin,axitinib, aflibercept, telatinib, brivanib alaninate, vatalanib,pazopanib, and ranibizumab;

Antibody drugs include, but are not limited to, trastuzumab, cetuximab,bevacizumab, rituximab, ticilimumab, ipilimumab, tremelimumab,pembrolizumab, nivolumab, Pidilizumab, RG-7446/ MPDL3280A, BMS-936559(MDX1105), Durvalumab (Medi-4736), MSB-0010718C, lumiliximab,catumaxomab, atacicept, oregovomab, panitumumab and alemtuzumab;

Antibody drugs also include antibody drug conjugates including but notlimited to those targeting Mesothelin, C4.4A, FGFR2, HER2, PSMA;

Antibody drugs also include Thorium targeted conjugates but not limitedto those targeting Mesothelin, C4.4A, FGFR2, HER2, PSMA, CEACAM6;

Antibody drugs also include bispecific (or multispecific) antibodyformats including but not limited to bispecific (or multispecific) IgGsand bispecific (or multispecific) antibody fragments as well as proteinfusions and conjugates thereof (e.g. CrossMab, DAF(2in1), DAF(4in1),DutaMab, DT-IgG, KiHassembled IgG, charge pair assembled IgG,KiH-commonLC, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, icX-mAb,orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv,scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG,KIH-IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (4in1),di-nanobody, BiTE, Diabody, DART, DART-Fc, TandAb, scDiabody,scDlabody-CH3, Diabody-CH3, Triple Body, Miniantibody, Minibody, TriBiminibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab′)2,F(ab′)2-scFv2, scFv- KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc,Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock fusion, ImmTAC,HSAbody, scDiabody-HAS, Tandem scFv-toxin, IgG-IgG, Cov-X-Body,scFv1-PEG-scFv2 and others);

Antibody drugs also include recombinant proteins generated byrecombinant technologies with antibody-like binding properties such asbut not limited to DARPIN molecules;

Cell therapies include, but are not limited to tumor infiltratinglymphocyte isolated from cancer patients such as Ex vivo stimulated Tcells e.g. Sipuleucel-T;

Cell therapies include, but are not limited to tumor infiltratinglymphocyte isolated from cancer patients such as Sipuleucel-T andgenetically engineered T cells bearing chimeric antigen receptors (CARs)such as e.g. CD19-CAR-T cells; CAR-Her2-T-cells;

Other anti-cancer agents including, e.g., alitretinoin, ampligen,atrasentan bexarotene, bortezomib, bosentan, calcitriol, exisulind,fotemustine, ibandronic acid, miltefosine, mitoxantrone, 1-asparaginase,procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin,tazaroten, velcade, gallium nitrate, canfosfamide, darinaparsin, andtretinoin, P13065, TG100-115;

EGFR (HER1) inhibitors such as, e.g., cetuximab, panitumumab, vectibix,gefitinib, erlotinib, and Zactima;

HER2 inhibitors such as, e.g., lapatinib, tratuzumab, and pertuzumab;

mTOR inhibitors such as, e.g., temsirolimus, sirolimus/Rapamycin, andeverolimus;

c-Met inhibitors;

Pi3K inhibitors such as PI3K inhibitor 1(2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamidedihydrochloride (see compound of Examples 1 and 2 WO 2012/136553)

and AKT inhibitors;

CDK inhibitors such as roscovitine and flavopiridol;

Spindle assembly checkpoints inhibitors and targeted anti-mitotic agentssuch as PLK inhibitors, Aurora inhibitors (e.g. Hesperadin), checkpointkinase inhibitors, and KSP inhibitors;

BRAFV600E inhibitors such as Vemurafenib, Dabrafenib;

HDAC inhibitors such as, e.g., panobinostat, vorinostat, MS275,belinostat, and LBH589;

HSP90 and HSP70 inhibitors;

Proteasome inhibitors such as bortezomib and carfilzomib;

Serine/threonine kinase inhibitors including MEK inhibitors and Rafinhibitors such as sorafenib;

Farnesyl transferase inhibitors such as, e.g., tipifarnib;

Tyrosine kinase inhibitors including, e.g., dasatinib, nilotibib,regorafenib, bosutinib, sorafenib, bevacizumab, sunitinib, cediranib,axitinib, aflibercept, telatinib, imatinib mesylate, brivanib alaninate,pazopanib, ranibizumab, vatalanib, cetuximab, panitumumab, vectibix,gefitinib, erlotinib, lapatinib, tratuzumab, pertuzumab, and c-Kitinhibitors;

Vitamin D receptor agonists;

Bcl-2 protein inhibitors such as obatoclax, oblimersen sodium, andgossypol;

Cluster of differentiation 20 receptor antagonists such as, e.g.,rituximab;

Ribonucleotide reductase inhibitors such as, e.g., gemcitabine;

Tumor necrosis factor related apoptosis inducing ligand receptor 1agonists such as, e.g., mapatumumab;

Tumor necrosis factor related apoptosis inducing ligand receptor 2agonists such as e.g., lexatumumab, conatumumab, CS-1008, PRO95780;

5-Hydroxytryptamine receptor antagonists such as, e.g., rEV598,xaliprode, palonosetron hydrochloride, granisetron, Zindol, and AB-1001;

Integrin inhibitors including alpha5-beta1 integrin inhibitors such as,e.g., E7820, JSM 6425, volociximab, and endostatin;

Androgen receptor antagonists including, e.g., nandrolone decanoate,fluoxymesterone, Android, Prost-aid, andromustine, bicalutamide,flutamide, apo-cyproterone, apo-flutamide, chlormadinone acetate,Androcur, Tabi, cyproterone acetate, and nilutamide;

Aromatase inhibitors such as, e.g., anastrozole, letrozole,testolactone, exemestane, aminoglutethimide, and formestane;

Matrix metalloproteinase inhibitors;

In addition, the antibodies of the invention can be combined withmodalities which cause immunogenic cell death including but not limitedto ultraviolet light, oxidizing treatments, heat shock, targeted anduntargeted radiotherapy, shikonin, high-hydrostatic pressure, oncolyticviruses, and photodynamic therapy;

In addition, the antibodies of the invention can be combined with agentswhich cause immunogenic cell death including but not limited tosunitinib, JAK2 inhibitors, anthracyclincs, doxorubicin, mitoxantrone,oxaliplatin, and cyclophosphamide, targeted and untargetedmicrotubule-destabilizing drugs (like e.g. auristatins andmaytansinoids);

The compounds of the present invention may also be employed in cancertreatment in conjunction with radiation therapy and/or surgicalintervention;

Furthermore, the antibodies of the invention may be utilized, as such orin compositions, in research and diagnostics, or as analytical referencestandards, and the like, which are well known in the art;

Diagnostic Methods

Anti-CEACAM6 antibodies or antigen-binding fragments thereof can be usedfor detecting the presence of CEACAM6-expressing tumors. The presence ofCEACAM6-containing cells or shed CEACAM6 within various biologicalsamples, including serum, and tissue biopsy specimens, may be detectedwith anti-CEACAM6 antibodies. In addition, anti-CEACAM6 antibodies maybe used in various imaging methodologies such as immunoscintigraphy witha ⁹⁹Tc (or other isotope) conjugated antibody. For example, an imagingprotocol similar to the one described using a ¹¹¹In conjugated anti-PSMAantibody may be used to detect pancreatic or ovarian carcinomas (Sodeeet al., Clin. Nuc. Med. 21: 759-766, 1997). Another method of detectionthat can be used is positron emitting tomography by conjugating theantibodies of the invention with a suitable isotope (see Herzog et al.,J. Nucl. Med. 34:2222-2226, 1993).

Pharmaceutical Compositions and Administration

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the present invention may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. An antibody of the invention or antigen-bindingfragment thereof can be administered by any suitable means, which canvary, depending on the type of disorder being treated. Possibleadministration routes include parenteral (e.g., intramuscular,intravenous, intra-arterial, intraperitoneal, or subcutaneous),intrapulmonary and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. In addition,an antibody of the invention or an antigen-binding fragment thereof or avariant thereof might be administered by pulse infusion, with, e.g.,declining doses of the antibody. Preferably, the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic. Theamount to be administered will depend on a variety of factors such asthe clinical symptoms, weight of the individual, whether other drugs areadministered. The skilled artisan will recognize that the route ofadministration will vary depending on the disorder or condition to betreated.

An embodiment of the present invention are pharmaceutical compositionswhich comprise anti-CEACAM6 antibodies or antigen-binding fragmentsthereof or variants thereof, alone or in combination with at least oneother agent, such as a stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. A furtherembodiment are pharmaceutical compositions comprising a CEACAM6 bindingantibody or antigen-binding fragment thereof and a furtherpharmaceutically active compound that is suitable to treat CEACAM6related diseases such as cancer. Any of these molecules can beadministered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In one embodimentof the present invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

The present invention also relates to the administration ofpharmaceutical compositions. Such administration is accomplished orallyor parenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co,Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl-cellulose, hydroxypropylmethylcellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores can be provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings forproduct identification or to characterize the quantity of activecompound, i.e. dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, andoptionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances that increase viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with acids, including but not limited to hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents that are the correspondingfree base forms. In other cases, the preferred preparation may be alyophilized powder in 1 mM-50 mM histidine or phosphate or Tris, 0.1%-2%sucrose and/or 2%-7% mannitol at a pH range of 4.5 to 7.5 optionallycomprising additional substances like polysorbate that is combined withbuffer prior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in an acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of anti-CEACAM6 antibodies orantigen-binding fragment thereof, such labeling would include amount,frequency and method of administration.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Therapeutically Effective Dose

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose, i.e. treatment of aparticular disease state characterized by CEACAM6 expression.

The determination of an effective dose is well within the capability ofthose skilled in the art.

Determining a therapeutically effective amount of the novel antibody ofthis invention or an antigen-binding fragment thereof or a variantthereof, largely will depend on particular patient characteristics,route of administration, and the nature of the disorder being treated.General guidance can be found, for example, in the publications of theInternational Conference on Harmonization and in REMINGTON'SPHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (18th ed.,Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). Morespecifically, determining a therapeutically effective amount will dependon such factors as toxicity and efficacy of the medicament. Toxicity maybe determined using methods well known in the art and found in theforegoing references. Efficacy may be determined utilizing the sameguidance in conjunction with the methods described below in theExamples.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., neoplastic cells, or inanimal models, usually mice, rabbits, dogs, pigs or monkeys. The animalmodel is also used to achieve a desirable concentration range and routeof administration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of antibody orantigen-binding fragment thereof, that ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors that may be taken into account include theseverity of the disease state, e.g., tumor size and location; age,weight and gender of the patient; diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered for example every 3 to 4 days, every week, onceevery two weeks, or once every three weeks, depending on half-life andclearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 10 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations forpolynucleotides than for proteins or their inhibitors. Similarly,delivery of polynucleotides or polypeptides will be specific toparticular cells, conditions, locations, etc. Preferred specificactivities for a radiolabelled antibody may range from 0.1 to 10 mCi/mgof protein (Riva et al., Clin. Cancer Res. 5:3275-3280, 1999; Ulaner etal., 2008 Radiology 246(3):895-902)

A further preferred embodiment of the invention is:

-   -   1. An isolated antibody or antigen-binding fragment thereof        specifically binding to human CEACAM6 and to Macaca fascicularis        CEACAM6.    -   2. An isolated antibody or antigen-binding fragment thereof        specifically binding to the mature extracellular domain of human        CEACAM6 (represented by amino acids at position 35-320 of SEQ-ID        No: 179) and to the mature extracellular domain of Macaca        fascicularis CEACAM6 (represented by amino acids at position        35-320 of SEQ-ID No: 177).    -   3. An isolated antibody or antigen-binding fragment thereof        specifically binding to human CEACAM6 domain 1 (represented by        amino acids 35-142 of SEQ-ID NO:179) and to Macaca fascicularis        CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID        NO:177).    -   4. An isolated antibody or antigen-binding fragment thereof        specifically binding to a protein comprising human CEACAM6        domain 1 (represented by amino acids 35-142 of SEQ-ID NO:179)        and to a protein comprising Macaca fascicularis CEACAM6 domain 1        (represented by amino acids 35-142 of SEQ-ID NO:177).    -   5. The antibody or antigen-binding fragment thereof according to        any one of embodiments 1 to 4, which does not significantly        cross-react with human CEACAM1, human CEACAM3, and human        CEACAM5.    -   6. The antibody or antigen-binding fragment thereof according to        any one of embodiments 1 to 5 able to bind to human and Macaca        fascicularis CEACAM6, or the mature extracellular domain        thereof, or the domain 1 thereof and the affinities are within        one order of magnitude of monovalent K_(D).    -   7. The antibody or antigen-binding fragment thereof according to        any one of the preceding embodiments which competes for binding        to the 9A6 antibody (Genovac/Aldevron) on human CEACAM6.    -   8. The antibody or antigen-binding fragment thereof according to        any one of the preceding embodiments which interferes with the        CEACAM6 and CEACAM1 interaction.    -   9. The antibody or antigen-binding fragment thereof according to        any one of the preceding embodiments which is able to change the        cytokine profile of tumor antigen specific T cells towards a        more activated phenotype characterized by an IFN-gamma secretion        increase, preferably by a≥1.5 times (1.5 times or higher)        increase compared to control samples.    -   10. The antibody or antigen-binding fragment thereof according        to any one of the preceding embodiments which is able to induce        immunomodulation.    -   11. The antibody or antigen-binding fragment thereof according        to any one of the preceding embodiments which is able to relieve        CEACAM6 mediated immunosuppression of tumor antigen specific T        cells as measured by either IFN-gamma secretion of tumor antigen        specific T cells or the number of IFN-gamma secreting activated        T cells.    -   12. The antibody or antigen-binding fragment thereof according        to any one of the preceding embodiments which is able to change        the cytokine profile of tumor antigen specific T cells towards a        more cytotoxic and/or activated phenotype characterized by an        IFN-gamma and/or IL-2 and/or TNF-alpha secretion increase,        preferably by a≥1.5 times (1.5 times or higher) increase of        IFN-gamma and/or IL-2 and/or TNF-alpha secretion compared to        control samples.    -   13. The antibody or antigen-binding fragment thereof according        to any one of the preceding embodiments which binds to an        epitope of human CEACAM6, wherein said epitope comprises one or        more amino acid residues selected from the group consisting of        Gln60, Asn61, Arg62, Ile63, Va183,Ile84, Gly85, Thr90, Ser127,        Asp128 and Leu129 of SEQ ID NO: 179.    -   14. The antibody or antigen-binding fragment thereof according        to embodiment 13 which binds to an epitope of human CEACAM6,        wherein said epitope comprises the amino acid residues Gln60,        Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128        and Leu129 of SEQ ID NO: 179.    -   15. The antibody or antigen-binding fragment thereof according        to embodiment 13 or 14 which binds to a human CEACAM6 protein        comprising an Ile63Leu mutation and which does not bind to a        human CEACAM6 protein comprising an Ile63Phe mutation according        to SEQ ID NO: 179 numbering.    -   16. The antibody or antigen-binding fragment thereof according        to any one of the preceding embodiments comprising:        -   i. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:48, an H-CDR2 comprising SEQ ID            NO:49, and an H-CDR3 comprising SEQ ID NO:50 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:52, a L-CDR2 comprising SEQ ID NO:53,            and a L-CDR3 comprising SEQ ID NO:54, or        -   ii. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:106, an H-CDR2 comprising SEQ ID            NO:107, and an H-CDR3 comprising SEQ ID NO:108 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:110, a L-CDR2 comprising SEQ ID NO:111,            and a L-CDR3 comprising SEQ ID NO:112, or        -   iii. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:4, an H-CDR2 comprising SEQ ID            NO:5, and an H-CDR3 comprising SEQ ID NO:6 and a light chain            antigen-binding region that comprises a L-CDR1 comprising            SEQ ID NO:8, a L-CDR2 comprising SEQ ID NO:9, and a L-CDR3            comprising SEQ ID NO:10, or        -   iv. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:34, an H-CDR2 comprising SEQ ID            NO:35, and an H-CDR3 comprising SEQ ID NO:36 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:38, a L-CDR2 comprising SEQ ID NO:39,            and a L-CDR3 comprising SEQ ID NO:40, or        -   v. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:120, an H-CDR2 comprising SEQ ID            NO:121, and an H-CDR3 comprising SEQ ID NO:122 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:124, a L-CDR2 comprising SEQ ID NO:125,            and a L-CDR3 comprising SEQ ID NO:126, or        -   vi. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:24, an H-CDR2 comprising SEQ ID            NO:25, and an H-CDR3 comprising SEQ ID NO:26 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:28, a L-CDR2 comprising SEQ ID NO:29,            and a L-CDR3 comprising SEQ ID NO:30, or        -   vii. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:76, an H-CDR2 comprising SEQ ID            NO:77, and an H-CDR3 comprising SEQ ID NO:78 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:80, a L-CDR2 comprising SEQ ID NO:81,            and a L-CDR3 comprising SEQ ID NO:82, or        -   viii. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:134, an H-CDR2 comprising SEQ ID            NO:135, and an H-CDR3 comprising SEQ ID NO:136 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:138, a L-CDR2 comprising SEQ ID NO:139,            and a L-CDR3 comprising SEQ ID NO:140, or        -   ix. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:148, an H-CDR2 comprising SEQ ID            NO:149, and an H-CDR3 comprising SEQ ID NO:150 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:152, a L-CDR2 comprising SEQ ID NO:153,            and a L-CDR3 comprising SEQ ID NO:154, or        -   x. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:14, an H-CDR2 comprising SEQ ID            NO:15, and an H-CDR3 comprising SEQ ID NO:16 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:18, a L-CDR2 comprising SEQ ID NO:19,            and a L-CDR3 comprising SEQ ID NO:20, or        -   xi. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:62, an H-CDR2 comprising SEQ ID            NO:63, and an H-CDR3 comprising SEQ ID NO:64 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:66, a L-CDR2 comprising SEQ ID NO:67,            and a L-CDR3 comprising SEQ ID NO:68, or        -   xii. a heavy chain antigen-binding region that comprises an            H-CDR1 comprising SEQ ID NO:92, an H-CDR2 comprising SEQ ID            NO:93, and an H-CDR3 comprising SEQ ID NO:94 and a light            chain antigen-binding region that comprises a L-CDR1            comprising SEQ ID NO:96, a L-CDR2 comprising SEQ ID NO:97,            and a L-CDR3 comprising SEQ ID NO:98.    -   17. The antibody or antigen-binding fragment thereof according        to any one of the preceding embodiments comprising:        -   i. a variable heavy chain sequence as presented by SEQ ID            NO: 47 and a variable light chain sequence as presented by            SEQ ID NO: 51, or        -   ii. a variable heavy chain sequence as presented by SEQ ID            NO: 105 and a variable light chain sequence as presented by            SEQ ID NO: 109, or        -   iii. a variable heavy chain sequence as presented by SEQ ID            NO: 3 and a variable light chain sequence as presented by            SEQ ID NO: 7, or        -   iv. a variable heavy chain sequence as presented by SEQ ID            NO: 33 and a variable light chain sequence as presented by            SEQ ID NO: 37, or        -   v. a variable heavy chain sequence as presented by SEQ ID            NO: 119 and a variable light chain sequence as presented by            SEQ ID NO: 123, or        -   vi. a variable heavy chain sequence as presented by SEQ ID            NO: 23 and a variable light chain sequence as presented by            SEQ ID NO: 27, or        -   vii. a variable heavy chain sequence as presented by SEQ ID            NO: 75 and a variable light chain sequence as presented by            SEQ ID NO: 79, or        -   viii. a variable heavy chain sequence as presented by SEQ ID            NO: 133 and a variable light chain sequence as presented by            SEQ ID NO: 137, or        -   ix. a variable heavy chain sequence as presented by SEQ ID            NO: 147 and a variable light chain sequence as presented by            SEQ ID NO: 151, or        -   x. a variable heavy chain sequence as presented by SEQ ID            NO: 13 and a variable light chain sequence as presented by            SEQ ID NO: 17, or        -   xi. a variable heavy chain sequence as presented by SEQ ID            NO: 61 and a variable light chain sequence as presented by            SEQ ID NO: 65, or        -   xii. a variable heavy chain sequence as presented by SEQ ID            NO: 91 and a variable light chain sequence as presented by            SEQ ID NO: 95.    -   18. The antibody according to any one of the preceding        embodiments, which is an IgG antibody.    -   19. The antibody according to embodiment 18 comprising:        -   i. a heavy chain region corresponding to SEQ ID NO: 57 and a            light chain region corresponding to SEQ ID NO: 58, or        -   ii. a heavy chain region corresponding to SEQ ID NO: 115 and            a light chain region corresponding to SEQ ID NO: 116, or        -   iii. a heavy chain region corresponding to SEQ ID NO: 43 and            a light chain region corresponding to SEQ ID NO: 44, or        -   iv. a heavy chain region corresponding to SEQ ID NO: 129 and            a light chain region corresponding to SEQ ID NO: 130, or        -   v. a heavy chain region corresponding to SEQ ID NO: 85 and a            light chain region corresponding to SEQ ID NO: 86, or        -   vi. a heavy chain region corresponding to SEQ ID NO: 143 and            a light chain region corresponding to SEQ ID NO: 144, or        -   vii. a heavy chain region corresponding to SEQ ID NO: 157            and a light chain region corresponding to SEQ ID NO: 158, or        -   viii. a heavy chain region corresponding to SEQ ID NO: 71            and a light chain region corresponding to SEQ ID NO: 72, or        -   ix. a heavy chain region corresponding to SEQ ID NO: 101 and            a light chain region corresponding to SEQ ID NO: 102.    -   20. The antigen-binding fragment according to embodiments 1 to        17, which is an scFv, Fab, Fab′ fragment or a F(ab′)₂ fragment.    -   21. The antibody or antigen-binding fragment according to any        one of the preceding embodiments, which is a monoclonal antibody        or antigen-binding fragment.    -   22. The antibody or antigen-binding fragment according to any        one of the preceding embodiments, which is human, humanized or        chimeric antibody or antigen-binding fragment.    -   23. An antibody-drug conjugate, comprising an antibody or        antigen binding fragment thereof according to any one of the        embodiments 1 to 22.    -   24. An isolated nucleic acid sequence that encodes the antibody        or antigen-binding fragment according to any one of the        embodiments 1 to 22.    -   25. A vector comprising a nucleic acid sequence according to        embodiment 24.    -   26. An isolated cell expressing an antibody or antigen-binding        fragment according to any one of the embodiments 1 to 22 and/or        comprising a nucleic acid according to embodiment 24 or a vector        according to embodiment 25.    -   27. An isolated cell according to embodiment 26, wherein said        cell is a prokaryotic or an eukaryotic cell.    -   28. A method of producing an antibody or antigen-binding        fragment according to any one of the embodiments 1 to 22        comprising culturing of a cell according to embodiment 27 and        purification of the antibody or antigen-binding fragment.    -   29. An antibody or antigen-binding fragment according to any one        of the embodiments 1 to 22 or an antibody-drug conjugate        according to embodiment 23 for use as a medicament.    -   30. An antibody or antigen-binding fragment according to any one        of the embodiments 1 to 22 or an antibody-drug conjugate        according to embodiment 23 for use as a diagnostic agent.    -   31. An antibody or antigen-binding fragment according to any one        of the embodiments 1 to 22 or an antibody-drug conjugate        according to embodiment 23 for use as a medicament for the        treatment of cancer.    -   32. An antibody or antigen-binding fragment according to any one        of the embodiments 1 to 22 or an antibody-drug conjugate        according to embodiment 23 in the manufacture of a medicament        for the treatment of a disease.    -   33. An antibody or antigen-binding fragment according to any one        of the embodiments 1 to 22 or an antibody-drug conjugate        according to embodiment 23 in the manufacture of a medicament        for the treatment of cancer.    -   34. A pharmaceutical composition comprising an antibody or        antigen-binding fragment according to any one of the embodiments        1 to 22 or an antibody-drug conjugate according to embodiment        23.    -   35. A combination of a pharmaceutical composition according to        embodiment 34 and one or more therapeutically active compounds.    -   36. A method for treating a disorder or condition associated        with the undesired presence of CEACAM6, comprising administering        to a subject in need thereof an effective amount of the        pharmaceutical composition according to embodiment 34 or a        combination according to embodiment 35.    -   37. A process for the preparation of anti-CEACAM6 antibodies        specifically binding to human CEACAM6 and Macaca fascicularis        CEACAM6, which process comprises immunization of an animal,        preferentially a mouse, with a protein comprising cynomolgus        CECAM6 domain 1 represented by amino acids 35-142 of SEQ-ID        NO:177, determining the amino acid sequence of antibodies        specifically binding to human CEACAM6 and to cynomolgus CEACAM6,        followed optionally by humanization or generation of a chimeric        antibody, and recombinant expression of said antibodies.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Example 1 Monkey CEACAM6 Sequences & Tool Generation

An overview of protein sequences of antigens and reference compoundsused is provided in Table 2:

TABLE 2 Name, Protein-IDs and SEQ-IDs used in this study Name Protein-IDDescription SEQ-ID human CEACAM6 TPP-4639 full-length SEQ-ID NO: 179Macaca fascicularis TPP-4189 full-length SEQ-ID NO: 177 CEACAM6 humanCEACAM1 TPP-4185 full-length SEQ-ID NO: 173 human CEACAM3 TPP-4187full-length SEQ-ID NO: 175 human CEACAM5 TPP-4188 full-length SEQ-ID NO:176 human CEACAM8 TPP-4190 full-length SEQ-ID NO: 178 human CEACAM19TPP-4186 full-length SEQ-ID NO: 174 human CEACAM6 TPP-1436 extracellular(mature SEQ-ID NO: 162 form) (R&D Systems 3934- CM) human CEACAM1TPP-1437 extracellular (mature SEQ-ID NO: 163 form) (R&D Systems 2244-CM) human CEACAM3 TPP-2755 extracellular (mature SEQ-ID NO: 172 form)(Sino Biological Inc. 11933-H08H) human CEACAM5 TPP-1438 extracellular(mature SEQ-ID NO: 164 form) (R&D Systems 4128- CM) Macaca mulattaTPP-1306 extracellular (mature SEQ-ID NO: 161 CEACAM6-Xa-Fc-His form),fusion to Xa-Fc- His Human CEACAM6-Xa- TPP-1790 extracellular (matureSEQ-ID NO: 165 Fc-His form), fusion to Xa-Fc- His Human CEACAM6-TPP-1791 extracellular (mature SEQ-ID NO: 166 Dom1-MacMul-Xa-Fc- form),fusion to Xa-Fc- His His, Domain 1 replaced by corresponding Macacamulatta domain Human CEACAM6- TPP-1792 extracellular (mature SEQ-ID NO:167 Dom2-MacMul-Xa-Fc- form), fusion to Xa-Fc- His His, Domain 2replaced by corresponding Macaca mulatta domain Human CEACAM6- TPP-1793extracellular (mature SEQ-ID NO: 168 Dom3-MacMul-Xa-Fc- form), fusion toXa-Fc- His His, Domain 3 replaced by corresponding Macaca mulatta domainHuman CEACAM6 APP-320 Extracellular (mature form), obtained by cleavagewith Factor Xa of TPP-1790 Human CEACAM6- TPP-1794 Domain 1, fusion toSEQ-ID NO: 169 Domain 1-His His (expressed in E. coli) Macacafascicularis TPP-2443 extracellular (mature SEQ-ID NO: 170CEACAM6-Xa-Fc-His form), fusion to Xa-Fc- His Macaca fascicularisAPP-319 extracellular (mature CEACAM6 form), obtained by Factor Xacleavage of TPP-2443 Macaca fascicularis TPP-2452 Domain 1, fusion toSEQ-ID NO: 171 CEACAM6-Domain 1- Xa-Fc-His Xa-Fc-His Macaca fascicularisAPP-325 Domain 1, obtained by CEACAM6-Domain 1 Factor Xa cleavage ofTPP-2452 Neo201 (human IgG1) TPP-1173 based on SEQ-ID NO: 1 &US20130189268 SEQ-ID NO: 2 Neo201 (human IgG2) TPP-3688 based on SEQ-IDNO: 89 & US20130189268 SEQ-ID: 90 9A6 (mouse IgG1) TPP-1744 Based onGenovac/Aldevron (GM-0509) 9A6 (chimeric hIgG1) TPP-1745 Based onGenovac/Aldevron (GM-0509) 9A6 (chimeric hIgG2) TPP-3470 Based onGenovac/Aldevron (GM-0509)

Protein sequences for human CEACAMs were obtained from UniProtKB/TrEMBLdatabase: human CEACAM6 (P40199), human CEACAM1 (P13688), human CEACAM3(P40198), human CEACAM5 (P06731), human CEACAM8 (P31997), human CEACAM19(Q7Z692). The Macaca mulatta (rhesus monkey) protein sequence of CEACAM6was also available (F6YVW1). The Macaca fascicularis (cynomolgus monkey)protein sequence of CEACAM6 was deduced from publicly availablenucleotide sequences by a) applying common intron/exon splicing rules b)comparison to different monkey/primate protein sequences and c)conservation of genomic structure between human/primate/monkey.Cynomolgus CEACAM6 is represented by TPP-4189.

Recombinant extracellular domains of CEACAMs were obtained fromcommercial sources or produced in-house. To this end, the extracellulardomains were C-terminally appended with a Factor Xa cleavage site, ahuman IgG1 Fc fragment as well as a His Tag and expressed in HEK293cells using standard transient transfection procedures. Proteins werepurified from the cell culture supernatant via Protein-A and sizeexclusion chromatography. In cases, in which the Fc-part needed to beremoved, proteins were cleaved with Factor Xa according to themanufacturer's recommendations (e.g. Factor Xa Protease from HematologicTechnologies Inc. HTI No. HCXA-0060) and subsequently purified byProtein-A and size exclusion chromatography. In cases in whichbiotinylated proteins were needed, commercial biotinylation kits wereused (e.g. EZ-Link Amine-PEG3-Biotin from Pierce #21347) and degree ofbiotinylation was characterized by commercial kits (e.g. BiotinQuantitation Kit from Pierce #28005).

The single N-terminal domain 1 of human CEACAM6 was produced as 6x Hisfused protein construct in E.coli BL21 DE3 using pET28a vector(Novagen). After overnight induction with IPTG at 37° C., recombinantprotein was isolated and refolded from inclusion bodies. Prior torefolding, inclusion bodies were washed in Tris buffer pH 8.5 containing150 mM NaCl, 1 mM EDTA, 0.1% Tween20 and solubilized in the same buffercontaining 8 M urea and no detergent. The solution was diluted (1:10)slowly into 50 mM CHES pH 9.2 containing 500 mM arginine and incubatedat 4° C. for 16 h. Purification was achieved performing standardNickel-NTA chromatography and size exclusion chromatography in 30 mMTris buffer pH 8.5, 150 mM NaCl on Superdex 75.

The 9A6 murine IgG1 antibody (GM-0509) was obtained from Genovac andchimerized to human IgG1 and human IgG2. The basis of Neo201 proteinsequence as either human IgG1 or human IgG2 was US20130189268. Allantibodies were expressed in HEK293 cells using standard transienttransfection procedures and purified from the cell culture supernatantvia Protein-A and size exclusion chromatography.

Stable HeLa cell lines expressing different full-length humanCEACAM-receptors were generated. Therefore sequences of the followingreceptors were transfected: human CEACAM1 (TPP-4185), human CEACAM3(TPP-4187), human CEACAM5 (TPP-4188), human CEACAM6 (TPP-4639), humanCEACAM8 (TPP-4190), human CEACEAM19 (TPP-4186) or cynomolgus CEACAM6(TPP-4189). The HeLa cell line does not endogenously express any ofthese receptors on the surface as was confirmed by FAGS analysis, andsurface expression could only be detected after transfection of therespective CEACAM-receptor. Briefly, expression constructs were clonedinto UCOE-based vectors (EMD Millipore Corporation) and transfected inHeLa cells. After selection with hygromycin, suitable stable clones werescreened by Western blotting of total cell lysate as well as FACSstaining of cellular surface using suitable antibodies (human CEACAM1:#MAB22441 from R&D Systems; human CEACAM5: #MAB41281 from R&D Systems;human CEACAM6: #MAB3934 from R&D Systems; human CEACAM8: ab90294 fromabcam; human CEACAM19: #NBP1-70494 from Novus; human CEACAM3: AF4166from R&D Systems; cynomolgus CEACAM6: Neo201-hIgG1).

Example 2 Characterization of Immunomodulating 9A6-mIgG1 Antibody

9A6 antibody has been described in the literature as beingimmunomodulatory (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503). This antibody was characterized with regards to itsaffinity, its selectivity towards other human CEACAMs, itscross-reactivity to monkey CEACAM6, its specific binding to a certaindomain on CEACAM6, and its selectivity towards other human CEACAMs.

Affinity Measurements Using Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) experiments for quantitative bindinganalyses were performed either using a Biacore T100, Biacore T200 or aBiacore 4000 instrument (GE Healthcare Biacore, Inc.) equipped withSeries S Sensor Chips CM5 (GE Healthcare Biacore, Inc.). Binding assayswere carried out at 25° C. with assay buffer HBS-EP+(10 mM HEPES pH 7.4,150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20). Antibodies were capturedwith an anti-hlgG capture antibody covalently immobilized to the chipsurface via amine coupling chemistry. Reagents for amine coupling(1-ethyl-3-(3-d imethylaminopropyl) carbodiimide hydrochloride (EDC),N-hydroxysuccinimide (NHS), ethanolamine-HCl pH 8.5) were used from theAmine Coupling Kit (GE Healthcare, product code BR-1000-50). Anti-hlgG,anti-mlgG capture antibodies and immobilization buffer (10 mM sodiumacetate pH 5.0) were used from the Human Antibody Capture Kit (GEHealthcare, BR-1008-39) and the Mouse Antibody Capture Kit (GEHealthcare, BR-1008-38), respectively. The sensor chip surface wasactivated with a freshly prepared solution of 0.2 M EDC and 0.05 M NHSpassed over the chip surface for 420 s at a flow rate of 10 μl/min,followed by an injection of anti-hlgG or anti-mlgG capture antibody(dissolved to 25 μg/ml in immobilization buffer) for 180 s at a flowrate of 5 μl/min. Excess of activated groups were blocked with a 1 molarsolution of ethanolamine injected at a flow rate of 10 μl/min for 420 s.

CEACAM antigens were used as analyte to determine K_(D) values.Antibodies were captured for 20 s at a flow rate of 10 μl/min prior toeach analyte injection. For kinetic affinity determination variousconcentrations between 1.56 and 200 nM of human CEACAM1, human CEACAM3,human CEACAM5, human CEACAM6, cynomolgus CEACAM6, cynomolgusCEACAM6-domain 1 protein in assay buffer (see above) were injected overthe captured antibodies at a flow rate of 60 μl/min for 3 minutes andthe dissociation was monitored for 10 minutes.

Obtained sensorgrams were double-referenced, i.e. in-line reference cellcorrection followed by buffer sample subtraction. K_(D) values werecalculated based on the ratio of dissociation (k_(d)) and association(k_(a)) rate constants which were obtained by globally fittingsensorgrams with a first order 1:1 Langmuir binding model, implementedin the Biacore Evaluation Software Package (Biacore T100/T200/4000Evaluation Software, GE Healthcare Biacore, Inc.).

Sandwich Competition Experiments by SPR

Sandwich competition experiments by SPR were performed in a similarmanner as outlined above with minor modifications. First, each antibodyto be analyzed was covalently immobilized on the sensor surface viaamine coupling (for details see above). To check whether a differentantibody competes for binding to a certain CEACAM antigen, therespective antigen was captured by injection over the immobilizedantibody and the second antibody to be tested for competition wasimmediately injected subsequently. If the second antibody binds to theantigen bound by the first antibody (+), both antibodies do not showcompetition and vice versa, if no binding is observed by injection ofthe second antibody (−), both antibodies compete for a similar epitope.

Domain Mapping Studies Using ELISA

To elucidate specific epitope information, several chimeric domainconstructs were designed, expressed and purified. Briefly, the wild typehuman CEACAM6 sequence was C-terminally fused with a human IgG1 Fcfragment, expressed in HEK293 cells and purified from the supernatantvia Protein-A and size exclusion chromatography (TPP-1790). In order tocreate different domain chimeras, the human sequence of one singledomain was consequently replaced by the corresponding Macaca mulattasequence (F6YVW1) in the Fc-fused human CEACAM6. This created threedifferent domain chimeras hDom1-hDom2-mDom3 (TPP-1793),hDom1-mDom2-hDom3 (TPP-1792), and mDom1-hDom2-hDom3 (TPP-1791), togetherwith the wild-type Macaca mulatta Fc fusion construct as control(TPP-1306). In addition to the chimeras, the single domain 1 of humanCEACAM6 was produced as described above from E. coli (TPP-1794).

Mapping of the domain specificity of the antibodies was carried outusing an ELISA assay:

For ELISA analysis, the Fc-fused domain chimeras and the single domain 1were coated on Nunc MaxiSorb plates and blocked with SmartBlocksolution. After incubation with an IgG-concentration series (1 nM-1000nM) for 1 h, plates were washed with PBS/T. Analysis of the bound IgGsto the CEACAM6 domain constructs was achieved over detection viaAnti-Human IgG (Fab specific)-Peroxidase antibody (A0293, Sigma).Fluorescence detection was performed with AmplexRed (A12222, Invitrogen)following standard protocols.

ELISA-Based Binding Analysis

ELISAs were used to characterize the binding of antibodies to variousCEACAM paralogs and orthologs. Black 384-well plates were coated with 25μl/well of various CEACAM protein preparations at 2 μg/ml in coatingbuffer (Candor) for 1 h at 37° C. After one wash with PBS/0.05%Tween-20, wells were blocked with 100% Smart Block (Candor) for one hourat 37° C. After three washes with PBS/0.05% Tween-20, dilution series ofthe antibodies in PBS/0.05% Tween-20/10% Smart Block ranging from 2pg/ml to 2 ng/ml were added and the plates were incubated for 1 h atroom temperature. After three washes with PBS/0.05% Tween-20, anappropriate secondary antibody was added. For detection of proteins witha human Fc such as TPP-1173, an anti-human IgG HRP (Sigma A0170) wasused at 1:10.000 dilution. For detection of proteins with a mouse Fcsuch as TPP-1744 an anti-mouse IgG HRP (ThermoScientific 31432) was usedat 1:10.000 dilution. PBS/0.05% Tween-20/10% Smart Block was used asdilution buffer. The plates were incubated for 1 hour at roomtemperature. After three washes, the plates were developed with AmplexRed (Life Technologies) and fluorescence was read at an emissionwavelength of 590 nm. GraphPad Prism 6.0 software was used to calculateEC₅₀ values using four-parameter non-linear curve fit.

Results

To measure the monovalent affinity of 9A6 to human CEACAM6 and to assessits cross-reactivity towards monkey CEACAM6, SPR experiments wereconducted as outlined above. Results are shown in Table 3:

TABLE 3 SPR analysis: monovalent K_(D) (in nM) Recombinant Recombinanthuman macaca mulatta CEACAM6 (R&D CEACAM6 (TPP- Alias Systems; TPP-1436)1306) 9A6-mIgG1 TPP-1744 22 — Neo201-hIgG1 TPP-1173 10 23 “—”: nobinding detected

As shown, 9A6-mIgG1 can bind with high affinity (22 nM) to recombinanthuman CEACAM6. However, no binding to Macaca mulatta CEACAM6 wasdetected. For comparison, Neo201-hIgG1 was also tested. This antibodydisplayed high affinity binding to both human and monkey CEACAM6. Insummary, 9A6 displays high affinity binding to human CEACAM6 but it isnot cross-reactive to monkey CEACAM6.

To map the binding domain of 9A6 on CEACAM6, binding on differenthuman/monkey chimera was assessed by ELISA as outlined above. To be ableto compare to Neo201-hIgG1 (TPP-1173), 9A6 was chimerized to a humanIgG1 (TPP-1745). Results are shown in Table 4:

TABLE 4 Domain mapping analysis by ELISA binding assay 9A6- Neo201-Origin Origin Origin hIgG1 hIgG1 of do- of do- of do- (TPP- (TPP- TPP-#main 1 main 2 main 3 1745) 1173) hWT 1790 human human human + + DOM11791 M. human human − + MM mulatta DOM2 1792 human M. human + + MMmulatta DOM3 1793 human human M. + + MM mulatta hDOM1 1794 human — — + −MM WT 1306 M. M. M. − + mulatta mulatta mulatta “+” denotes bindingdetected; “−” denotes no binding detected

9A6 is able to bind to wildytpe human CEACAM6, and to chimeras employingDomain 2 or 3 of Macaca mulatta CEACAM6. However, it fails to bind to achimera employing Domain 1 of Macaca mulatta CEACAM6 or to wildtypeMacaca mulatta CEACAM6. Consistent with this, it is able to bind to thesingle domain 1 of human CEACAM6. In contrast, Neo201-hIgG1 binds to allforms tested except for single domain 1 of human CEACAM6. In conclusion,9A6 binds to the N-terminal Domain 1 of human CEACAM6.

To substantiate the results, a competition experiment was performed asoutlined above. Results are shown in Table 5.

TABLE 5 Sandwich competition experiments by SPR on recombinant humanCEACAM6 (R&D Systems, TPP-1436) 9A6-mIgG1 (TPP-1744) Neo201-hIgG1(TPP-1173) 9A6-mIgG1 (TPP-1744) − + Neo201-hIgG1 (TPP-1173) + − If thesecond antibody binds to the antigen bound by the first antibody (+),both antibodies do not show competition and vice versa, if no binding isobserved by injection of the second antibody (−), both antibodiescompete for a similar epitope

As evident from Table 5, 9A6-mIgG1 and Neo201-hIgG1 do not compete witheach other for binding to human CEACAM6. This is consistent with thepublished epitope of Neo201 residing outside domain 1.

To analyze the selectivity of 9A6 towards different CEACAM6 orthologsand to allow comparison to Neo201-hIgG1, 9A6 was chimerized to hIgG1(TPP-1745). In ELISA binding experiment conducted as outlined above, theEC₅₀ values listed in Table 6 have been obtained:

TABLE 6 Selectivity/cross-reactivity analysis by binding ELISA - EC₅₀values in nM Human Human Cynomolgus Human CEACAM6 CEACAM3 CEACAM5CEACAM6 TPP- (TPP-1436) (TPP-2755) (TPP-1438) (APP-319) 9A6-hIgG1TPP-1745 0.09 — — — Neo201- TPP-1173 0.11 — 0.09 1.35 hIgG1 “—” denotesEC₅₀ > 10 nM

High affinity binding of 9A6 to human CEACAM6 was confirmed. Consistentwith SPR experiments using rhesus monkey CEACAM6, 9A6 also fails to bindto cynomolgus monkey CEACAM6. It is, however, selective for CEACAM6since no binding to human CEACAM3 or CEACAM5 was observed.

In contrast, Neo201-hIgG1 displays similar high affinity binding tohuman CEACAM6 and is even cross-reactive to cynomolgus CEACAM6. Thiscomes at a reduced selectivity, since it displays also high affinitybinding to human CEACAM5.

Example 3 Protein Sequence Alignment of CEACAMs

The mature extracellular form of human CEACAM6 (amino acids 35-320 ofUniProtKB/Swiss-Prot: P40199.3) consists of different domains:N-terminal domain 1 (according to www.uniprot.org amino acids 35-142 ofUniProtKB/Swiss-Prot: P40199.3), domain 2 (according to www.uniprot.orgamino acids 145-232 of UniProtKB/Swiss-Prot: P40199.3), and domain 3(according to www.uniprot.org amino acids 237-314 ofUniProtKB/Swiss-Prot: P40199.3). Since the goal was to identify aselective, high affinity antibody to N-terminal domain 1 of CEACAM6, yetbeing cross-reactive to cynomolgus monkey CEACAM6, the probability ofcombining the desired properties in one molecule was assessed.

To this end, the protein sequence of N-terminal domain 1 of humanCEACAM6 was compared to other proteins using Blastp algorithm (NCBI)using standard settings to identify most relevant CEACAM6 homologs.(Partial) mature extracellular domains of human CEACAM6 (amino acids35-320 of UniProtKB/Swiss-Prot: P40199.3), human CEACAM1 (amino acids35-428 of UniProtKB/Swiss-Prot: P13688.2), human CEACAM3 (amino acids35-155 of UniProtKB/Swiss-Prot: P40198.2), human CEACAM5 (amino acids35-417 of UniProtKB/Swiss-Prot: P06731.3) and cynomolgus (macacafascicularis) CEACAM6 (amino acids 35-320 of TPP-4189) were alignedusing “Global Alignment—Wilbur and Lipman (fast)” in Phylosophersoftware (Genedata). The alignment is shown in FIG. 1. The percentagesequence identities of N-terminal domainl of human CEACAM6 to otherN-terminal domains (according to alignment) were determined using VectorNTI Software (Life Technologies). Those results are shown in Table 7.

TABLE 7 Percentage of protein sequence identities of N-terminal domainsof different CEACAMs to N-terminal domain 1 of human CEACAM6. Sequenceidentity of N-terminal domains to N-terminal domain 1 of human CEACAM6Human CEACAM6 (100%)  Human CEACAM3 90% Human CEACAM1 90% Human CEACAM589% Cynomolgus CEACAM6 81%

The sequence alignment in FIG. 1 shows a very high degree of similarityof protein sequences of human CEACM6 and human CEACAM3, human CEACAM5and human CEACAM1 throughout the entire extracellular region. The targetregion (domain 1 of human CEACAM6) is especially similar to otherCEACAMs, which is also reflected in Table 7. The paralogs of humanCEACAM6 are much more similar to human CEACAM6 than the cynomolgusortholog. In fact, there are only 2 positions in N-terminal region inthe primary sequence that are identical in human and cynomolgus CEACAM6but different from amino acids in this position in the other humanparalogs (marked in FIG. 1 with asterisks).

To conclude: it is highly challenging to identify a high affinityantibody to the N-terminal domain 1 of human CEACAM6 that is selectivebut still cross-reactive to monkey CEACAM6.

Example 4 Antibody Generation by Phage Display

To identify human anti-CEACAM6 antibodies, various selections with thehuman Fab-phage library FAB-300 from DYAX were performed, essentially asdescribed earlier (Hoet et al., 2005; Huang et al., 2006). As summarizedin Table 8, different strategies with up to 4 rounds of phage selectionwere employed using biotinylated Fc-tagged recombinant CEACAM6 fromhuman and cynomolgus monkey (TPP-1436 & TPP-2443), coated onstrepavidin-beads, and the human tumor cell line KPL-4 (Kurebayashi etal., Br J Cancer. 1999 February; 79 (5-6):707-17), which is expressinghigh amounts of endogenous target protein on the cell surface. Inaddition, depletion for binder against CEACAM5 (TPP-1438; hC5) andCEACAM1 (TPP-1437; hC1) (off-targets) or recombinant human IgG1-Fc (Fc)was included as indicated prior to each selection on protein targets.For example, in strategy A after depletion on human CEACAM1-coated beads(hC1), the first round of panning was done on human CEACAM6 (hC6). Theresulting output was divided and one part was used for a second and athird round of selection on human CEACAM6. The other part was used forsecond round of panning on KPL-4 cells, a third round on human CEACAM6and a final fourth panning round on KLP-4 cells. In strategy C, aspecific elution step was performed, using the mouse mAb 9A6-mIgG1(TPP-1744).

TABLE 8 Phage selection strategies: human hC6 = TPP- 1790; cynomolguscynoC6 = TPP-2443, hC1 = TPP-1438, hC5 = TPP-1437, Fc = RecombinantHuman IgG1 Fc (R&D Systems #110-HG-100) Strategy: A B C D Round 1 hC6hC6 hC6 cynoC6 Round 2 hC6 KPL-4 hC6 KPL-4 hC6 KPL-4 hC6 Round 3 hC6 hC6hC6 hC6 hC6 hC6 cynoC6 Round 4 — KPL-4 — KPL-4 — KPL-4 hC6 Depletion hC1hC1 and hC5 hC1 Fc Specific — — 9A6 — elution

Phage pools enriched from different rounds of selections were screenedfor binders to target and off-target by Fab-phage ELISA as described(Hoet et al., Nat Biotechnol. 2005 March; 23(3):344-8) or byFACS-analysis on CEACAM6-expressing cells. Phage pools with a favorableprofile were selected for genelll-removal and subsequent ELISA-screeningof soluble Fabs in an ELISA. DNA of resulting sFab-hits was sequencedand unique representatives characterized for cell-binding byFACS-analysis on KPL-4 cells (Table 9). In some strategies phage binderaccording to the invention were directly re-cloned into IgG.

TABLE 9 FACS-titration of unique sFab-hits: Protein-ID FACS-titration assFab on KPL-4 (as hIgG1) cells TPP-1667 ++ TPP-1668 + TPP-1669 +TPP-1670 + TPP-1672 + TPP-1673 + TPP-1674 + TPP-1676 + TPP-1677 0TPP-1678 0 TPP-1679 +++ TPP-1680 + TPP-1684 0 TPP-1686 + +++: >1000events @ ~2.2 μg/ml sFab ++: >100 events @ ~2.2 μg/ml sFab +: >100events @ ~6.7 μg/ml sFab 0: below threshold

Binding of phage display selected, purified Fab fragments (see list inTable 9) to biotinylated variants of human CEACAM1 (TPP-1437), humanCEACAM5 (TPP-1438) and human CEACAM6 (TPP-1436) was analyzed by biolayerinterferometry using an Octet RED384 instrument (Pall ForteBio Corp.).Biotinylated antigens were loaded onto Streptavidin (SA) Biosensors(ForteBio Part number 18-5019) and after a baseline equilibrium step inassay buffer (PBS supplemented with 0.1% (w/v) BSA, 0.02% (v/v) Tween20and 0.05% (v/v) sodium azide; ForteBio Part number 18-5032), binding ofFabs diluted in assay buffer to a final concentration of 200 nM wasmonitored for 300 seconds followed by a dissociation phase of 300seconds.

The corresponding purified Fab fragments from Table 9 also displayedbinding to human CEACAM6 but not to human CEACAM5 or human CEACAM1.

In order to analyze whether the Fabs compete with 9A6 for binding tohuman CEACAM6, a competition experiment was carried out. Here,biotinylated human CEACAM6 (TPP-1436) was loaded onto SA Biosensors andbinding responses of Fabs were compared to binding responses of Fabsobtained with loaded CEACAM6 saturated with 9A6-mIgG1 (TPP-1744) (asoutlined in Example 1). If the binding response in presence of 9A6 issignificantly reduced or abolished this is a strong indication that atested Fab binds to an epitope similar to that of 9A6.

Surprisingly, all Fabs tested were able to compete with 9A6 for bindingto human CEACAM6.

The Fab sequences were reformatted into human IgG1 format for furthercharacterization.

Affinities (monovalent K_(D)) of reformatted antibodies towardsrecombinant human CEACAM6 (TPP-1436) were determined by SPR analogouslyto experimental procedures described in Example 2. Sensorgrams wereeither evaluated by globally fitting sensorgrams with a first order 1:1Langmuir binding model or with a steady-state affinity analysisimplemented in the Biacore Evaluation Software (Biacore T200/4000Evaluation Software) Package. Results are shown in Table 10:

TABLE 10 SPR analysis: monovalent K_(D) (in nM) Protein-ID K_(D) (in nM)TPP-1667 (750) TPP-1668 (550) TPP-1669 (185) TPP-1670 (340) TPP-1672(580) TPP-1673 (515) TPP-1674 (600) TPP-1676 (1980)  TPP-1677 (580)TPP-1678 (300) TPP-1679  76 TPP-1680 (870) TPP-1684 (480) TPP-1686 (310)TPP-2968 weak values in brackets: not accurately determined underpresent experimental conditions but are sufficient for comparison amongeach other

As evident from Table 10, the antibodies displayed rather low monovalentaffinities, the lowest value (highest affinity) being 76 nM forTPP-1679. Three IgGs displaying the highest monovalent affinities wereanalyzed with regards to their selectivity and cross-reactivity tocynomolgus CEACAM6 in an ELISA binding experiment (carried out inanalogy to the protocol given in Example 2)

TABLE 11 Selectivity/cross-reactivity analysis by binding ELISA: EC₅₀values in nM Human Human Human Cynomolgus CEACAM6 CEACAM3 CEACAM5CEACAM6 TPP- (TPP-1436) (TPP-2755) (TPP-1438) (APP-319) TPP-1679 0.162.24 0.86 — TPP-1669 0.22 — 1.01 — TPP-1678 0.19 — — 42.74 “—” denotesEC₅₀ > 10 nM (in the case of cynomolgus CEACAM6 > 100 nM)

To summarize, antibodies with rather poor monovalent affinities havebeen obtained. This might be a trade-off due to avoiding binding toother paralogs. Still, selectivity profile is often insufficient (seeTPP-1679 & TPP-1669 in Table 11). From all antibodies tested, TPP-1678is the only one with a very marginal cross-reactivity to recombinantcynomolgus CEACAM6 (see Table 11).

In conclusion, no therapeutically useful anti-CEACAM6 antibodies havebeen obtained using phage display without further maturation.

Example 5 Antibody Maturation of Phage Display-Derived Antibodies

To obtain antibodies with desirable affinity, selectivity andcross-reactivity profiles, some phage display-derived antibodies wereaffinity-matured.

Therefore, all CDR amino acid positions of TPP-1669, TPP-1678 andTPP-1679 were randomized individually. The resulting variants wereexpressed and assessed for binding to multiple CEACAM family members(human CEACAM6, cynomolgus CEACAM6, human CEACAM3 and human CEACAM5) bybinding ELISA in cell supernatants.

For TPP-1669, individual mutations enhancing binding to cynomolgusCEACAM6 without at the same time enhancing binding to other human CEACAMfamily members as well could not be identified.

For TPP-1679, several individual mutations were identified, whichenhanced binding to human CEACAM6 without concomitant enhanced bindingto other human CEACAM family members, and a recombination librarycontaining all possible permutations was generated. The correspondingvariants were expressed as human IgG2 isotypes, purified and assessedfor binding to multiple CEACAM family members by SPR analogously toexperimental procedures described in Example 2. Table 12 summarizes theproperties of selected antibodies obtained by this process.

TABLE 12 SPR analysis: monovalent K_(D) (in nM) Human Cynomolgus HumanHuman CEACAM6 CEACAM6 CEACAM5 CEACAM3 Human CEACAM1 (R&D (APP-319; (R&D(Sino (R&D Systems; (TPP-2443 Systems; Biological; Systems; TPP-1436)cleaved) TPP-1438) TPP-2755) TPP-1437) TPP-3399  9 — weak weak —TPP-3400  6 weak weak — — TPP-3401 16 —  (94) — — TPP-3402 18 — (172) —— TPP-3403 13 weak (144) — — TPP-3404 11 (363) (138) — — TPP-3405 14(522) (126) weak — TPP-3406 (15) — — — — TPP-3407 (12) — — weak —TPP-3408 (27) — — — — values in brackets: not accurately determinedunder present experimental conditions “—”: no binding detected undercurrent experimental conditions “weak”: if at most the two highestanalyte concentrations analyzed (i.e. 100 and 200 nM) resulted in abinding signal that is between three times the signal to noise ratio and20 percent of the theoretical maximum binding response(Rmax_(theoretical))

The results obtained in Table 12 indicate that affinity and selectivityenhancement is very well possible. However, it also underscores thechallenge to obtain cynomolgus CEACAM6 cross-reactive binders, which areat least within one order of magnitude close to the monovalent affinitytowards human CEACAM6.

For TPP-1678, several individual mutations were identified, whichenhanced binding to human CEACAM6 and cynomolgus CEACAM6 without greaterconcomitant enhanced binding to other human CEACAM family members, and arecombination library containing several permutations was generated. Thecorresponding variants were expressed as human IgG2 isotypes, purifiedand assessed for binding to multiple CEACAM family members by SPRanalogously to experimental procedures described in Example 2(summarized in Table 13).

The binding characteristics of these antibodies were also determined bya binding ELISA (monovalent binding, biotinylated CEACAM proteins): A1:440 dilution of of an anti-human IgG (Sigma, 12136) in Coating buffer(Candor) was used to coat black 384-well Maxisorp plates (Nunc) for 1hour at 37° C. After one wash with PBS/0.05% Tween-20 the plates wereblocked with 100% SmartBlock (Candor) for 1 hour at 37° C. After threewashes, 2 μg/ml of the relevant antibodies were added to the plate inPBS/0.05% Tween-20/10% SmartBlock. The plates were incubated for 1 hourat room temperature. After three washes dilution series of the relevantbiotinylated CEACAM proteins in PBS/0.05% Tween-20/10% SmartBlock wereadded and the plates were incubated for one hour at room temperature.After three washes 1 μg/ml of Streptavidin-Peroxidase (Sigma, S5512) inPBS/0.05% Tween-20/10% SmartBlock was added and the plates wereincubated for 30 minutes at room temperature. After three washes, theplates were developed with Amplex Red (Life Technologies) andfluorescence was read at an emission wavelength of 590 nm. GraphPadPrism 6.0 software was used to calculate EC₅₀ values usingfour-parameter non-linear curve fit.

Variants with a substantially improved affinity, selectivity andcross-reactivity profile were identified as summarized in Table 13 andTable 14 for selected antibodies obtained by this process.

TABLE 13 SPR analysis: monovalent K_(D) (in nM) Human Cynomolgus HumanHuman CEACAM6 CEACAM6 CEACAM5 CEACAM3 Human CEACAM1 (R&D (APP-319; (R&D(Sino (R&D Systems; (TPP-2443 Systems; Biological; Systems; TPP-1436)cleaved) TPP-1438) TPP-2755) TPP-1437) TPP-3705 28 32 — — — TPP-3707 2832 — — — TPP-3708 3 5 — weak (158) TPP-3709 8 20 — — (408) values inbrackets: not accurately determined under present experimentalconditions “—”: no binding detected under current experimentalconditions “weak”: if at most the two highest analyte concentrationsanalyzed (i.e. 100 and 200 nM) resulted in a binding signal that isbetween three times the signal to noise ratio and 20 percent of thetheoretical maximum binding response (Rmax_(theoretical))

TABLE 14 Selectivity/cross-reactivity analysis by binding ELISA: EC₅₀values in nM Human Human Human CEACAM3 Human CEACAM5 CEACAM6 CEACAM1(Sino (R&D (R&D Cynomolgus (R&D Biological Systems Systems CEACAM6Systems Inc. TPP- TPP- TPP-1436 (APP-319- TPP-1437 2755 1438 TPP-biotinylated) biotinylated) biotinylated) biotinylated) biotinylated)TPP-3705 0.18 0.12 ~3.82 × 10⁶ 113.04 — (ambiguous fit) TPP-3707 0.190.11 ~8.94 × 10⁵ 139.18 — (ambiguous fit) TPP-3708 0.02 0.03  9.64 1.04— TPP-3709 0.04 0.07 36.53 4.68 — TPP-3470 0.08 — — — — (9A6-hIgG2) “—”:denotes no binding detectable up to highest concentrations tested (150ng/ml for human and cynomolgus CEACAM6; 2000 ng/ml for human CEACAM1,human CEACAM3, human CEACAM5)

In conclusion, using the TPP-1678 precursor, it was possible to obtainfew high affinity human antibodies to human CECACAM6 that are trulycross-reactive to cynomolgus CEACAM6 and that are selective to CEACAM6:binding of TPP-3707 to human CEACAM6 is about 730-fold better than tohuman CEACAM3 (620-fold for TPP-3705) as judged by comparing theircorresponding EC₅₀ values.

Example 6 Antibody Generation by Mouse Immunization

To generate mouse monoclonal antibodies against CEACAM6, two differentimmunization strategies were performed based on the sequence ofimmunogens applied to the Balb/c mice (strategy A and B in Table 15).Within each strategy, mice were immunized either via footpad orintraperitoneal application of antigens over 5 rounds of injection, asdepicted in Table 15. Strategy A was focusing on the immunization withcynomolgus CEACAM6-Domainl, whereas Strategy B was based on thecombination of full-length extracellular CEACAM6 from human andcynomolgus monkey as immunogens.

Immunisations by footpad were based on 5 injections of 1 μg antigen onceweekly. Immunizations by intra-peritoneal route were based on 4 IPinjections biweekly (10 μg of antigen) followed by one boost byintravenous injection.

TABLE 15 Immunization schedule Strategy A Stategy B FootpadIntraperitoneal Footpad Intraperitoneal immunization immunizationimmunization immunization 1^(st) injection cynoC6-D1 cynoC6-D1 hC6(APP-320) hC6 (APP-320) (APP-325) (APP-325) 2^(nd) injection cynoC6-D1cynoC6-D1 CynoC6 (APP- CynoC6 (APP- (APP-325) (APP-325) 319) 319) 3^(rd)injection cynoC6-D1 cynoC6-D1 hC6 (APP-320) hC6 (APP-320) (APP-325)(APP-325) 4^(th) injection cynoC6-D1 cynoC6-D1 CynoC6 (APP- CynoC6 (APP-(APP-325) (APP-325) 319) 319) 5^(th) injection cynoC6-D1 cynoC6- hC6(APP-320) + hC6 (APP-320) + (APP-325) D1*(APP-325) CynoC6 (APP- CynoC6(APP- 319) 319)* *i.v. boost

Four days after the last injection, lymph nodes or spleen cells of micewere fused according to standard methods (e.g. Kohler and MilsteinNature. 1975 Aug. 7; 256(5517):495-7). Screening of resultinghybridoma-clones was done in an ELISA using biotinylated antigens andoff-target proteins (as listed in Table 16). In more detail,microtiter-plates were coated with goat anti-mouse antibodies overnightat 4° C. The following day plates were washed and blocked with 5% BSAfor 2 h at room temperature, followed by another washing step. 20 μl ofhybridoma supernatants were incubated with biotinylated antigens for 1 hat room temperature and the mixtures transferred to the coated wellsfollowed by an incubation step (1 h at room temperature). After washingthe plates, anti-streptavidin-HRP conjugates were added for 30 min atroom temperature. Finally, wells were washed, and the color reaction wasdeveloped by addition of 50 μl TMB and recorded in a plate reader.

TABLE 16 List of proteins used in ELISA-screening of hybridomas (targetand off-targets) Target (all biotinylated) Off-target (all biotinylated)Human CEACAM-6 (TPP-1436) hCEACAM1 (TPP-1437) Cynomolgus CEACAM6-Fc(TPP-2443) hCEACAM5 (TPP-1438) Cynomolgus CEACAM6 (APP-319) Fc cleaved

Surprisingly, only Strategy A resulted in clones showing a favorableprofile with regards to human & cynomolgus CEACAM6 cross-reactivity aswell as selectivity. In addition, some species specific clones wereobtained from both strategies.

Candidates selected positively by ELISA were subcloned over at least 3cloning rounds and produced at larger amounts from ascites fluid byprotein A chromatography.

Antibodies from mouse immunizations were also characterized for bindingto CEACAM6 in a cellular context. HeLa-cells overexpressing human orcynomolgus CEACAM6 were employed in FACS experiments with eithersupernatants from hybridoma or purified mlgs (see Example 1).Non-transfected HeLa-cells served as negative control. Table 17summarizes the profile of identified candidates from ELISA andFACS-analysis:

TABLE 17 Summary of qualitative results for binding of murinehybridoma-derived antibodies to human and cynomolgus CEACAM6 from ELISA(using biotinylated TPP-1436, TPP-2443 and APP-319) and FACS-analysis(using transfected HeLa cells see Example 1: TPP-4639 and TPP-4189)Protein-ID Human CEACAM6 Cynomolgus CEACAM6 TPP-2969 no bindingdetectable binding TPP-2970 no binding detectable binding TPP-2971binding binding TPP-3100 binding binding TPP-3187 binding bindingTPP-3101 binding binding TPP-3186 binding binding

The murine antibodies obtained were characterized more closely withregards to their monovalent affinities (K_(D)), their selectivitytowards other human paralogs and their degree of cross-reactivity tocynomolgus CEACAM6 by SPR analysis as purified mlgGs.

SPR was conducted analogously to experimental procedures described inExample 2.

Results are summarized in Table 18:

TABLE 18 SPR analysis: monovalent K_(D) (in nM) Cynomolgus HumanCynomolgus CEACAM6 Human Human Human CEACAM6 CEACAM6 Domain 1 CEACAM5CEACAM3 CEACAM1 (R&D (APP-319; (APP-325; (R&D (Sino (R&D Systems;(TPP-2443 TPP-2452 Systems; Biological; Systems; TPP-1436) cleaved)cleaved) TPP-1438) TPP-2755) TPP-1437) TPP-2969 72 (109)  51 − − −TPP-2970 72 56 47 − − − TPP-2971 61 25 30 − − − TPP-3100 79 n.t. 52 − −n.t. TPP-3101 69 n.t. 13 (300) weak n.t. TPP-3186 74 n.t. 53 − − n.t.TPP-3187 68 n.t. 42 − − n.t. values in brackets: not accuratelydetermined under present experimental conditions “−”: no bindingdetected under current experimental conditions “weak”: if at most thetwo highest analyte concentrations analyzed (i.e. 100 and 200 nM)resulted in a binding signal that is between three times the signal tonoise ratio and 20 percent of the theoretical maximum binding response(Rmax_(theoretical)) n.t—not tested

There were unresolved discrepancies for TPP-2969 & TPP-2970 observed: ininitial ELISA and FAGS analysis they appeared cynomolgus CEACAM6specific, whereas in later SPR experiment they exhibited also binding torecombinant human CEACAM6.

To summarize: immunization of mice with cynomolgus CEACAM6 N-terminaldomain 1 (APP-325) suprisingly yielded some antibodies (e.g. TPP-3186,TPP-2971, TPP-3187) that are truly human—cynomolgus CEACAM6cross-reactive and at the same time selective with regards to otherhuman paralogs. The affinities are in an acceptable range fortherapeutic puposes, yet their murine origine and associatedimmunogenicities preclude therapeutic applications in humans.

Example 7 Antibody Humanization

To generate antibodies suitable for therapeutic applications in humans,selected murine antibodies were humanized.

Selected sequences of the murine hybridoma-derived antibodies weredetermined by sequencing the antibody cDNAs of the respective hybridomacell lines (see Table 17). According to the sequencing results, TPP-3100and TPP-3186 are identical. TPP-3101 yielded a single heavy chain buttwo light chain sequences. TPP-2971 and TPP-3187 were highly similar.They differed in four amino acids (see FIG. 2).

The deciphered murine VH and VL sequences of the antibody TPP-2971 andTPP-3187 were humanized by grafting the CDRs according to the Kabatdefinition into human germline frameworks. As an exception, HCDR2 waspartially grafted. Since this CDR is very long (16 amino acids)according to the Kabat definition, only the first 9 amino acids weregrafted. These amino acids represent the part of HCDR2 which isidentical to HCDR2 according to the Chothia definition (for CDRdefinitions according to Kabat and Chothia see: Andre C. R. Martin,“Protein sequence and structure analysis of antibody variable domains”in Antibody Engineering (Springer Lab Manuals), Eds.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg).

The human germline frameworks were chosen based on similarity searchesof the murine framework pieces FW1, FW2, FW3, and FW4 with the set ofhuman VH and VL as well as J element germline sequences. The murine CDRswere grafted into the best matching germline sequences (excluding theCDRs), which were IGKV1-9*01 and IGKJ2*01 for VL (Identities 69.6%, FW1;86.7%, FW2; 71.9%, FW3; 80.0%, FW4) and IGHV2-70*01 and IGHJ6*01 for VH((Identities: 73.3% (TPP-2971) and 70.0% (TPP-3187), FW1; 85.7%, FW2;71.9%, FW3; 90.9%, FW4)). Germline sequences applied in similaritysearches were derived from the VBASE2 data set (Retter I, Althaus H H,Munch R, Muller W: VBASE2, an integrative V gene database. Nucleic AcidsRes. 2005 Jan. 1; 33(Database issue):D671-4). The names assigned to themost similar germline sequences were taken from the IMGT system(Lefranc, M.-P., Giudicelli, V., Ginestoux, C., Jabado-Michaloud, J.,Folch, G., Bellahcene, F., Wu, Y., Gemrot, E., Brochet, X., Lane, J.,Regnier, L., Ehrenmann, F., Lefranc, G. and Duroux, P. IMGT®, theinternational ImMunoGeneTics information system®. Nucl. Acids Res, 37,D1006-D1012 (2009); doi:10.1093/nar/gkn838)).

Two variants of humanized sequences derived from TPP-2971 have beengenerated: TPP-3310 and TPP-3714. In VH of TPP-3310 the J element waskept unchanged compared to the murine originator, whereas in VH ofTPP-3714 the J element was made completely human germline-like (see FIG.3). No glycosylation sites or unpaired cysteines were found in thehumanized sequences.

In addition, two variants of humanized sequences derived from TPP-3187were generated: TPP-3820 and TPP-3821 (see FIG. 4). In comparison toTPP-3714, VH of TPP-3820 contained threonine instead of serine at theposition 30 in HFW1, and glycine instead of alanine at position 46 inHFW2, while VL contained two asparagine residues instead of two serineresidues at positions 92 and 93 in LCDR3. These four amino acidexchanges reflect the differences between the murine originatorsequences of TPP-3187 and TPP-2971.

The variable domain VH of TPP-3821 is identical to TPP-3714, while VLcontained two asparagine residues instead of two serine residues atpositions 92 and 93 in LCDR3 in comparison TPP-3714. These two aminoacid exchanges reflect the differences in the CDRs between the murineoriginator sequences of TPP-3187 and TPP-2971. No glycosylation sites orunpaired cysteine residues were found in the sequences of TPP-3820 andTPP-3821.

Affinity determination and sandwich competition experiments ofchimerized and humanized antibodies were performed by SPR analogously toexperimental procedures described in Example 2 and the results aresummarized in Table 19 and Table 20:

TABLE 19 Sandwich competition experiments by SPR on recombinant humanCEACAM6 (R&D Systems, TPP-1436) TPP-3308 TPP-3322 TPP-3323 (hIgG2 (hIgG2(hIgG2 TPP-3470 chimera chimera chimera (hIgG2 of of of chimeraTPP-2971) TPP-3186) TPP-3187) of 9A6) TPP-3308 − − − − (hIgG2 chimera ofTPP-2971) TPP-3322 − − − − (hIgG2 chimera of TPP-3186) TPP-3323 − − − −(hIgG2 chimera of TPP-3187) TPP-3470 − − − − (hIgG2 chimera of 9A6 Ifthe second antibody binds to the antigen bound by the first antibody(+), both antibodies do not show competition and vice versa, if nobinding is observed by injection of the second antibody (−), bothantibodies compete for a similar epitope

TABLE 20 SPR analysis: monovalent K_(D) (in nM) Human Cynomolgus HumanHuman Human CEACAM6 CEACAM6 CEACAM5 CEACAM3 CEACAM1 (R&D (APP-319; (R&D(Sino (R&D Systems; (TPP-2443 Systems; Biological; Systems; TPP-1436)cleaved) TPP-1438) TPP-2755) TPP-1437) TPP-3310 13 31 − − − TPP-3714 1327 − − − TPP-3820 27 54 − − − TPP-3821 24 49 − − − “−” denotes nobinding detected under current experimental conditions

Selectivity and cross-reactivity analysis was carried out by bindingELISA (monovalent, biotinylated CEACAM proteins) in analogy to theprotocol provided in Example 5. Results obtained are summarized in Table21.

TABLE 21 Selectivity/cross-reactivity analysis by binding ELISA: EC₅₀values in nM Human Human Human CEACAM3 Human CEACAM6 CEACAM1 (SinoCEACAM5 (R&D Cynomolgus (R&D Biological (R&D Systems CEACAM6 SystemsInc. TPP- Systems TPP-1436 (APP-319- TPP-1437 2755 TPP-1438 TPP-biotinylated) biotinylated) biotinylated) biotinylated) biotinylated)TPP-3310 0.09 0.07 − − − TPP-3714 0.09 0.07 − − − TPP-3470 0.08 − − − −(hIgG2 chimera of 9A6 “−” denotes no binding detectable up to highestconcentrations tested (150 ng/ml for human and cynomolgus CEACAM6; 2000ng/ml for human CEACAM1, human CEACAM3, human CEACAM5)

The results in Table 19 indicate TPP-2971, TPP-3186 & TPP-3187 competefor the same or a similar epitope on human CEACAM6 as 9A6-hIgG2. Theresults in Table 20 and Table 21 underscore a high affinity binding tohuman & cynomolgus CEACAM6 with true crossreactivity while beingselective to CEACAM6 and not binding to CEACAM6 paralogs.

In conclusion, humanization was fully successful, with antibodiesexhibiting even higher affinities than their murine precursors, enablinga therapeutic application in humans.

Example 8 Selective CEACAM6 Binding on Cells

To demonstrate binding and selectivity of the anti-CEACAM6 antibodies toauthentic antigens, the antibodies were tested for binding to nativeCEACAM6 on the cell surface of different cell lines by FACS experiments.

CEACAM6 selectivity was tested on a panel of HeLa-cells which have beentransfected with different CEACAM-receptors (human CEACAM1, humanCEACAM3, human CEACAM5, human CEACAM6, human CEACAM8, human CEACAM19 andcynomolgus CEACAM6 - see Example 1) in comparison to binding of HeLawild type cells which were shown to be CEACAM6 negative. EG50 valueswere determined for the binding to human and cynomolgus monkey CEACAM6transfected HeLa cells. Results are shown in Table 22.

For FACS experiments HeLa wild type cells were cultured in RPMI-1640,10% FCS, while CEACAM-receptor transfected HeLa cells received inaddition 0.5% Gentamycin (stock 10 mg/ml, Fa. PAA) and 200 μg/mlHygromycin B (stock 50 mg/ml, Invitrogen). Cells were washed 3 timeswith PBS w/o Ca²⁺/Mg²⁺ and were detached from the culture platenon-enzymatically with EDTA dissociation buffer (Gibco). Cells werewashed in cold FACS buffer (PBS w/o Ca²⁺/Mg²⁺ and heat-inactivated 3%FCS) and were counted using a countess machine (Invitrogen). 10⁵ cellsper well were plated and incubated with the respective primary antibody(5 μg/ml) for 1 h at 4° C. on a plate shaker. Then cells were washed(400 g, 5′) with FACS buffer 2 times, were resuspended in 100 μlcontaining the secondary antibody (PE-anti-mouse or anti-human IgG,1:150 dilution, Dianova #115-115-164, #109-115-098) and were incubatedfor another 1 h at 4° C. on a plate shaker. After 2 times washing thecells were resuspended in 100 μl FACS buffer and were analysed on a FACSCanto II machine (Beckton Dickinson) or a FACS Array (BecktonDickinson).

For EC₅₀ analysis, the primary antibodies were used at increasingconcentrations in a range from 0.1 nM to 100 nM. Half maximal bindingvalues (EC₅₀) were determined by plotting the median fluorescenceintensity signal against the concentration (logarithmic scale). Curvefitting of data was performed using the Graph Pad prism analysissoftware.

TABLE 22 Specific binding to CEACAM-receptor transfected HeLa cell linepanel. HeLa HeLa human Cynomolgus HeLa HeLa HeLa CEACAM6 HeLa HeLaCEACAM6 HeLa human human human TPP-4639 human human TPP-4189 TestSpecies, wild CEACAM1 CEACAM3 CEACAM5 EC₅₀ CEACAM8 CEACAM19 EC₅₀antibody isotype type TPP-4185 TPP-4187 TPP-4188 [nM] TPP-4190 TPP-4186[nM] TPP-2971 mIgG1 − − − + − ++ 0.5 0.5 TPP-3100 mIgG1 − − − ++ − ++ 0.35 3 TPP-3186 mIgG1 − − − ++ − ++ 0.5 0.5 TPP-3187 mIgG1 − − − ++ −++  0.35 4 TPP-3322 Hu/mIgG1 − − − − + − ++ 0.6 0.8 TPP-3323 Hu/mIgG1 −− − − + − ++ 0.6 0.8 TPP-3308 Hu/mIgG1 − − − − + − ++  0.45 0.6 TPP-3820hIgG2 − − − − +++ − − 1 (+) 1   TPP-3821 hIgG2 − − − − +++ − − 1 (+) 1  TPP-3310 hIgG2 − − − − +++ − − 1.5 (+) 0.6 TPP-3714 hIgG2 − − − − +++ −− 1 (+) 1   TPP-3707 hIgG2 − + + − +++ − − 4 19   TPP-3470 hIgG21   >100 (9A6- hIgG2) Anti-CEACAM 6 mIgG1 − − − − +++ − − clone 9A6(Genovac #GM0509) Definition of −, +, ++, +++ as determined from FACSmedian fluorescence log shift: − = no shift; + = log shift as comparedto control antibody, median 10-100; ++ = 2 log shift, median 100-1,000;+++ = 3 log shift, median 1,000-10,000

Anti-CEACAM6 antibodies were also tested for their binding to differentcancer cell lines that endogenously express CEACAM6 by FAGS analysis.Cell lines were cultured according to the protocols provided by theAmerican tissue culture collection (ATCC). The observed binding signalwas specific as the non-binding isotype control did not result into ashift of the fluorescence signal. Half maximal binding (EC50) values arein the low nanomolar range (Table 23).

TABLE 23 Binding of anti-CEACAM6 antibodies to endogenously CEACAM6positive tumor cell lines (EC₅₀ values) TPP-3470 TPP-3310 (9A6-hIgG2)EC50 EC50 Cell line* [nM] [nM] PaTu-8902  0.15 0.2 SNU-C1 0.5 0.8 KS 0.81.0 NCI-H1993 0.8-1.6 1.6 T84 0.3 0.35 *All cell lines from publictissue bank such as the American tissue culture collection etc, exceptKS breast cancer cell line kindly provided by Dr. Brigitte Giickel(University of Tübingen).

In conclusion, for the murine TPP-2971, TPP-3100, TPP-3186 & TPP-3187antibodies as well as the human TPP-3820, TPP-3821, TPP-3310, TPP-3714 &TPP-3707 antibodies, selective binding to human authentic cell-surfaceCEACAM6 was demonstrated (no binding to other human paralogs). Thebinding of these antibodies to human CEACAM6 is comparable to 9A6 onhuman CEACAM6 expressing cell lines. For TPP-3310, a similar binding as9A6-hIgG2 to endogenously expressed CEACAM6 on human tumor cell lineswas demonstrated.

Antibodies of the invention also bind to cynomolgus CEACAM6 with acomparable avidity as to the human receptor in the single-digit tosubnanomolar binding EC₅₀ range while no binding of 9A6 to thecynomolgus CEACAM6 on the cell surface was detected up to 100 nM. Thisresult indicates a true crossreactivity to human and cynomolgus CEACAM6for the antibodies of the invention while 9A6 clearly bindspreferentially to human CEACAM6.

Example 9 Thermal Stability Analysis

Thermal stability of IgGs was investigated using Differential Scanningcalorimetry (DSC) using a VP-Capillary DSC system (MicaCal Inc.) with acell volume of 0.137 mL. All samples were diluted in DPBS pH.7.4 to afinal concentration of 0.5 mg/mL and a buffer control without proteinwas used as a reference. The samples were scanned from 20° C. to 120° C.at a scan rate of 120° C./h. Resulting thermograms were corrected bysubtraction of buffer control scans and normalized for proteinconcentration using Origin 7.0 Data analysis (OriginLab Corp.). Meltingtemperatures were obtained by fitting the DSC data to a nonlinearregression routine (“Non-2-state: Cursor init”) provided with Origin.

TABLE 24 Thermal Stability of Fab domain Thermal Stability of Fab domainTPP-3310 88.2° C. TPP-3714 88.5° C.*/95.1° C.* TPP-3400 74.7° C.TPP-3707 80.6° C. TPP-3470 80.2° C. (9A6-hIgG2) *Fab unfoldsnon-cooperatively

All of the IgGs measured display a high thermal stability within the Fabdomain. The thermal stability of TPP-3310 and TPP-3714 is remarkablyhigh and exceeding by far the thermal stability of TPP-3470 (9A6-hIgG2).This is surprising since high stability has been associated withantibodies possessing VH3 framework (Honegger et al., 2009, Protein EngDes Sel. 22(3):121-134).

High thermal stability is indicative of a better pharmaceuticalsuitability of TPP-3310 and TPP-3174 as compared to TPP-3470 (9A6-hIgG2)(better stability, less propensity to aggregation, less risk ofimmunogenicity).

Example 10 Interference with Interaction Between CEACAM6 and CEACAM1

It has been hypothesized that CEACAM1 might be the binding partner forCEACAM6 in trans on activated T cells (Witzens-Harig et al., Blood 2013May 30; 121(22):4493-503): trans and cis homophilic and heterophilicinteractions amongst CEACAMs have been described, for example betweenCEACAM1 and CEACAM5 or CEACAM6 and CEACAM8. CEACAM1 is displayed onactivated T cells, CEACAM1 ligation and phosphorylation recruitsSH2-domain-containing protein tyrosine phosphatase 1 (SHP1). SHP1dephosphorylates ZAP70, which results in the inhibition of TCRsignaling. Thereby, CEACAM1 ligation leads to an early inhibition ofT-cell activation within 10 minutes after activation.

Moreover, a role for CEACAM5 in the inhibition of natural killer (NK)cell responses against colorectal cancer cells was reported (Zheng etal., PLoS One. 2011; 6(6):e21146), which might be based on itsheterophilic binding to inhibitory CEACAM1 expressed on the NK cells.

Therefore, a direct CEACAM6-CEACAM1 interaction was tested usingrecombinant proteins in a binding ELISA. After establishing inpreliminary experiments that a moderate but specific interaction betweenCEACAM1 and CEACAM6 could be detected, the following protocol was used:Black 384-well Maxisorb plates (Nunc) were coated with 1 μg/ml CEACAM1(R&D Systems, TPP-1437) in Coating Buffer (Candor) for 1 h at 37° C. orwere left uncoated as a control. After one wash with PBS/0.05% Tween-20the wells were blocked with 100% Smart Block (Candor) for 1 h at 37° C.In separate plates, dilution series of antibodies of interest inPBS/0.05% Tween-20, 10% SmartBlock were incubated with 2 μg/mlCEACAM6-Fc (TPP-1790) for 1 h at RT. The blocked plates were washedthree times and the preformed antibody-CEACAM6-complexes were added. Theplates were incubated for 1 h at RT. After three washes an anti-humanIgG HRP (Sigma A1070) was added at 1:10.000 and the plates wereincubated for 1 h at RT. After three washes the plates were developedwith Amplex Red (Life Technologies) and fluorescence was read at anemission wavelength of 590 nm.

TABLE 25 Competition ELISA for antibodies competing with binding ofhuman CEACAM6-Fc (TPP-1790) to passively coated human CEACAM1(TPP-1437). Competition with binding of human CEACAM6-Fc to humanCEACAM1 TPP-3400 + TPP-3310 + TPP-3714 + TPP-3323 + TPP-3705 +TPP-3707 + TPP-3470 + (9A6-hIgG2) TPP-3688 − (Neo201-hIgG2) “+” denotescompetition “−” denotes no competition

As shown in Table 25, it was possible to compete the interaction ofCEACAM6 with CEACAM1 with all antibodies tested except for TPP-3688(Neo201-hIgG2).

In conclusion, this observation is consistent with the hypothesis of a)CEACAM1 on activated T cells being a possible interaction partner forCEACAM6 leading to inhibition of T cells, b) the N-terminal D1 domain ofCEACAM6 being implicated in the interaction between CEACAM1 and CEACAM6,and c) antibodies of the invention being capable to interfere withCEACAM6-CEACAM1 interaction.

Example 11 Inhibition of Immunosuppressive Activity of CEACAM6 In Vitro

The immunosuppressive function of CEACAM6 on tumor cells was studiedrecently in vitro (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503) and in vivo (Khandelwal et al., Poster Abstract 61,Meeting Abstract from 22nd Annual International Cancer ImmunotherapySymposium Oct. 6-8, 2014, New York City, USA). Commercially available9A6 antibody (Genovac/Aldevron) was shown to be able to inhibitimmunosuppressive activity of CEACAM6, leading to enhanced cytokinesecretion by T cells in vitro and anti-tumor efficacy in vivo.

To study the effect of the antibodies of the invention on theimmunosuppressive activity of CEACAM6, co-culture experiments of a modeltumor cell line with a model tumor antigen-specific T cell clone wereconducted:

Tumor-antigen specific T cells were generated by a procedure describedin Brackertz et al (Brackertz et al., Blood Cancer J. 2011 March;1(3):e11). Briefly, survivin specific CD8⁺ T cells were isolated fromperipheral mononuclear cells via CD8-specific magnetic-activated cellsorting. The isolated HLA-A2⁻CD8⁺ T cells were repetitively stimulatedwith allogenic HLA-A2⁺ dendritic cells loaded with 10 μg of theHLA-restricted peptide epitope Survivin₉₅₋₁₀₄ (ELTLGEFLKL). Afterstimulation, the proliferating T cells were stained withHLA-A2/Survivin₉₅₋₁₀₄ multimers (A*02:01 391 LMLGEFLKL Survivin 96-104labeled with APC, Prolmmune Limited, #F391-4A-E), FACS sorted and clonedby limiting dilution in 96-well plates.

The T cell clone expansion was performed by culturing 2×10⁵ T cellclones and feeder cells composed of 5×10⁷ irradiated PBMCs (30 Gy) and1×10⁷ irradiated LCL (these B lymphoblastoid cell lines that weregenerated by EBV transduction of peripheral blood B cells from healthydonors with a EBV-infected monkey cell line (B95/8, ATTC), as describedin Brackertz et al., Blood Cancer J. 2011 March; 1(3):e11 andDissertation Andreas Moosmann, Ludwig-Maximilians-University Munich,Germany, 2002) from different donors (100-150 Gy) in 40 ml of RPMI-1640medium with glutamine (Sigma-Aldrich), 10% human serum (Human AB serum,Valley Biomedical, Inc, #HP1022), 1% Penicillin/Streptomycin (LifeTechnologies) at 37° C. and 5% CO₂. The expansion occurred in thepresence of 50 U/ml IL-2 (Proleukin, Novartis, #1003780), 2.5 ng/mlIL-15 (rhIL-15-CF R&D #247_IL-025/CF) and 30 ng/ml anti-human CD3antibody (OKT3 eBiosciences 16-0037-85) for 14 days. The KS human breastcancer cell line (obtained from Dr. Brigitte Gückel (University ofTubingen, Germany)) was cultured in DMEM (Sigma-Aldrich) with 10% FCS(FBS Superior, Biochrom) and 1% Penicillin/Streptomycin at 37° C. and 5%CO₂.

To analyze the modulatory activity of the anti-CEACAM6 antibodies on theimmunosuppressive function of CEACAM6 in vitro, the survivin-peptidespecific CD8⁺ T cell clone was co-cultivated together with the CEACAM6⁺,HLA-A2⁺ and survivin⁺ human breast cancer cell line KS and IFN-gammasecretion as readout for T cell activity was measured either byIFN-gamma ELISpot or IFN-gamma ELISA.

For the co-culture, KS tumor cells were detached non-enzymatically usingPBS-EDTA for 5 min, centrifuged, washed and counted. Cell concentrationwas adjusted to 1×10⁵ cells/ml in X-Vivo-20 (Lonza) and cells werepretreated with anti-CEACAM6 antibodies or isotype-matched controlantibodies for 10 min on ice. After the incubation step, 10,000 KStarget cells were seeded directly in triplicates to IFN-gamma-ELISpot orU-96-Well ELISA plates, respectively. In the meantime, survivin-peptidespecific T cells were harvested, washed with X-Vivo-20 and seeded in thecell numbers indicated on the KS target cells. The co-culture of tumorcells, anti-CEACAM6 antibodies and T cells was incubated for 20-40 h at37° C. IFN-gamma ELISpot plates (MABTECH: ELISpot Assay for humanInterferon gamma #3420-3PT, Antibodies mAB 1-D1K anti-IFNg, mAB7-B6-1-Biotin, Steptavivin-ALP, BCIP/NBT plus substrate for ELISpot#3650-10) and IFN-gamma-ELISA (BD human IFN-gamma ELISA Set #555142)were developed according to the manufacturer's instructions. ELISpotplates were counted with a C.T.L. ELISpot plate reader and opticaldensity for ELISA plates was measured with a Tecan Infinite M200 platereader. An experiment was considered as valid if the positive controlTPP-3470 (9A6-hIgG2) was statistically significant compared to theisotype-matched antibody control.

Co-culture of KS tumor cells with survivin-peptide specific CD8⁺ T cellsin the presence of anti-CEACAM6 antibodies resulted in a statisticallysignificant increase of IFN-gamma production by the T cells (FIG. 5)compared to the samples not treated with anti-CEACAM6 antibody ortreated with isotype-matched control antibody.

In conclusion, cynomolgus cross-reactive antibodies TPP-3310, TPP-3707,and TPP-3323 were able to relieve CEACAM6 mediated immunosuppression oftumor antigen specific T cells to the same extent as TPP-3470(9A6-hIgG2) as measured by either IFN-gamma secretion ofsurvivin-peptide specific CD8⁺ T cells or number of IFN-gamma secretingactivated T cells.

Example 12 Analysis of Cytokine/Chemokine Profile Secreted by T CellsTreated with Anti-CEACAM6 Antibodies

In order to study the effects of anti-CEACAM6 antibodies on the human Tcell cytokine/chemokine profile towards an improved cytotoxicity and aneffective anti-tumor immune response, Luminex-based multiplex cytokineanalysis of co-culture experiments of a model tumor cell line and amodel tumor antigen specific T cell clone were performed.

A survivin-peptide specific CD8⁺ T cell clone was generated and expandedin vitro as described in Example 11. Tumor cell culture and ELISAco-culture were performed as described in Example 11.

After 20 h of co-culture plates were centrifuged for 10 min at 1400 rpmand supernatant was collected. Multiplex analysis was performed usingthe MILLIPLEX Human Cytokine/Chemokine Magnetic Bead Panel - Premixed 38Plex analytes (Merck Millipore #HCYTMAG-60K-PX38) on a BioPlex100 System(Bio-Rad) according to manufacturer's instructions. Standard curves andconcentrations were calculated with Bio-Plex Manager 6.0. An experimentwas considered as valid if the positive control TPP-3470 (9A6-hIgG2)was >1.5× increased compared to the isotype-matched antibody control.

Blockade of CEACAM6 by antibodies of the invention in the co-culture ofsurvivin-peptide specific T cells with KS tumor cells yields a >1.5times increase in IFN-gamma, IL-2 and TNF-alpha secretion compared tothe control samples that were treated with the isotype-matched control(FIG. 6).

In conclusion, the cynomolgus cross-reactive antibodies TPP-3310 andTPP-3707 are able to change the cytokine profile of survivin-peptidespecific CD8⁺ T cells towards a more cytotoxic and activated phenotypecharacterized by increased IFN-gamma, IL-2 and TNF-alpha secretion asmeasured by Luminex-based multiplex analysis to the same extent asTPP-3470 (9A6-hIgG2).

Example 13 Anti-Tumor Efficacy in Adoptive T Cell Transfer KS Model

The anti-tumor efficacy of an anti-CEACAM6 antibody (9A6;Genovac/Aldevron) has been studied in vivo in adoptive human T celltransfer systems in which tumor-antigen specific human T cells areexpanded in vitro and co-injected with an anti-CEACAM6 antibody intonude mice bearing human xenograft tumors (Khandelwal et al., PosterAbstract 61, Meeting Abstract from 22nd Annual International CancerImmunotherapy Symposium Oct. 6-8, 2014, New York City, USA).

To study the effect of antibodies of the invention on anti-tumorefficacy, the following adoptive T cell transfer experiment wasconducted:

A survivin-peptide specific CD8⁺ T cell clone was generated and expandedin vitro as described in Example 11.

Six to eight weeks old female NOD-Scid mice (NOD.CB17-Prkdc^(scid)/J;Charles River, France) were injected subcutaneously with 2×10⁶ KS tumorcells (see Example 11). Randomization of mice was performed on day 16and mice with a tumor surface lower than 40 mm² were excluded (=no tumortake). Mice (n=8-10 mice per group) were treated on day 23 and 27 withi.v. adoptive transfer of 5×10⁶ survivin-peptide specific T cell clone.200 μg of the anti-CEACAM6 antibodies TPP-3740, TPP-3707, TPP-3310 orthe respective isotype-matched control antibody were administered i.p.on days 22, 24, 26 and 28. The control group was injected with PBSinstead of T cells and antibodies. Subcutaneously grown tumors weremeasured with a caliper and the surface was then calculated by using theformula “length×width”. Only experiments were considered as valid, inwhich the vehicle-treated control group of mice exhibited a steady andsignificant increase of tumor surface and tumor volume throughout theentire duration of the study. The outcome of the experiment might beinfluenced by parameters that are difficult to control: not only the invivo growth of KS cell lines proved to be variable but also survival ofhuman T cells in mice as well T cell infiltration into tumors exhibitedconsiderable variation.

Adoptive transfer of survivin-peptide specific T cells in combinationwith the anti-CEACAM6 antibodies tested resulted in a reduced tumorburden compared to T cells injected with the matched isotype control orthe PBS control group (FIG. 7). Similar efficacy was observed usingTPP-3740 (9A6-hIgG2).

In conclusion, cynomolgus cross-reactive antibodies TPP-3310 andTPP-3707 exhibited anti-tumor efficacy to the same extent as TPP-3470(9A6-hIgG2) in an adoptive T cell transfer model using survivin-peptidespecific CD8⁺ T cells and KS tumors.

Example 14 Tumor Cell Lines and Tumor Tissues that are CEACAM6 Positive

CEACAM6 is expressed in various cancers which are potential targetindications for treatment with CEACAM6 immunomodulating antibodies.Therefore cancer cell lines of different origin and which representdifferent cancers were tested for CEACAM6 expression by FACS analysis.The results are shown in Table 26.

Cancer cell lines which were acquired from public tissue banks such asthe American tissue culture collection (ATCC) etc. were culturedaccording to the provider's instructions.

Cells were washed 3 times with PBS w/o Ca²⁺/Mg²⁺ and were detached fromthe culture plate non-enzymatically with EDTA dissociation buffer(Gibco). Cells were washed in cold FACS buffer (PBS w/o Ca²⁺/Mg²⁺ andheat-inactivated 3% FCS) and were counted using the cell countercountess machine (Invitrogen). 10⁵ cells per well were plated andincubated with the mouse monoclonal antibody 9A6 (TPP-1744; 5 μg/ml) orthe purified NA/LE Mouse IgG1 Isotype control antibody (BD Pharmingen#553447) for 1 h at 4° C. on a plate shaker. Then cells were washed (400g, 5′) with FAGS buffer 2 times, were resuspended in 100 μl containingthe PE-labeled anti-mouse secondary antibody (1:150 dilution, Dianova#115-115-164) and were incubated for another 1 h at 4° C. on a plateshaker. After 2 times washing the cells were resuspended in 100 μl FACSbuffer and were analyzed on a FAGS Canto II machine (Beckton Dickinson)or a FACS Array (Beckton Dickinson). The observed binding signal wasspecific as the non-binding isotype control did not result into a shiftof the fluorescence signal (Table 26).

TABLE 26 Result of human cancer cell line screening by FACS for bindingof CEACAM6 specific antibody 9A6 mIgG1 (TPP-1744) and thus CEACAM6expression CEACAM6 Cancer surface Cell line* Origin Type expression MCF7breast adenocarcinoma + MCF7/AdrVp (MDR breast adenocarcinoma + breastCA) KS breast malignant effusion, breast cancer +++ patient BT-20 breastadenocarcinoma, ER negative − BT-474 breast ductal carcinoma; +++ HCC38breast primary ductal carcinoma − MDA-MB-361 breast adenocarcinoma,derived from +++ metastatic site: brain MDA-MB-453 breast metastaticcarcinoma, derived − from metastatic site: pericardial effusion MFM-223breast epithelial-like, ductal carcinoma +/− MX-1 breast infiltratingductal carcinoma, ER− − negative SUM 149 breast invasive ductalcarcinoma, ER− − PR− inflammatory breast cancer SUM 159 breast Er−, PR−anaplastic carcinoma of − the breast T-47D breast ductal carcinoma, frommetastatic + site: pleural effusion ZR-75-1 breast ductal carcinoma,from metastatic ++ site: ascites HPAC pancreas adenocarcinoma ++++SW1990 pancreas adenocarcinoma, from metastatic − site: spleen PaTu 8902pancreas adenocarcinoma +−++ AsPC-1 pancreas adenocarcinoma, frommetastatic +++ site: ascites DAN-G pancreas adenocarcinoma + MIA PaCa-2pancreas − Panc1 pancreas ductal, epithelioid pancreatic −− carcinomaBxPc3 pancreas adenocarcinoma +++ Capan-2 pancreas adenocarcinoma −PC3.gd.neo prostate adenocarcinoma, from metastatic − (PaCa) site: boneHPAFII (PaCa) pancreas adenocarcinoma +++ SW 1463 rectum epithelials,Dukes' type C, +/− colorectal adenocarcinoma SNU-C1 colon adencarcinoma,from metastatic ++ site: peritoneum HT55 colon epithelial +++ Colo 201colon Dukes type D, adenocarcinoma, − from metastatic site: ascitesColo320DM colon Dukes type D, colorectal − adenocarcinoma CaCo-2 colonadenocarcinoma +−++ SW403 colon Dukes type C adenocarcinoma − HCC2998colon adenocarcinoma ++ RKO colon − KM-12 colon adenocarcinoma ++ CL-34large adenocarcinoma + intestine COLO 205 colon Dukes type D,adenocarcinoma ‘+/− HCT 116 colon − SW480 colon Duke type B, colorectal− adenocarcinoma WiDr colon adenocarcinoma ++ DLD-1 colon Dukes Type C,adenocarcinoma − SW 620 colon Dukes Type C, adenocarcinoma, − frommetastatic site: lymph node SW1116 colon adenocarcinoma +++ T-84 coloncolon metatases from the lung +−++ LoVo colon adenocarcinoma ++ HCT15colon Dukes type C, adenocarcinoma − HT29 colon adenocarcinoma ++ LS174Tcolon Dukes type B, adenocarcinoma +/− EKVX lung adenocarcinoma − A549lung +++ NCI-H1688 lung SCLC + Calu-1 lung epidermoid carcinoma, K-ras −positive Calu-3 lung adenocarcinoma + HCC827 lung adenocarcinoma ++LXF-289 lung adenocarcinoma − NCI-H1299 lung from metastatic site: lymphnode − NCI-H1437 lung adenocarcinoma, from metastic +++ site. pleuraleffusion NCI-H146 (SCLC) lung derived from metastatic site (bone +/−marrow) NCI-H1581 lung Large cell NSCLC − NCI-H1975 lung adenocarcinoma− NCI-H1993 lung adenocarcinoma, from metastatic +++ site: lymph nodeNCI-H2228 lung adenocarcinoma ++ NCI-H226 lung squamous cell carcinoma,− mesothelioma, from metastatic site: pleural effusion NCI-H23 lungadenocarcinoma − NCI-H292 lung mucoepidermoid pulmonary − carcinomaNCI-H322 lung bronchioalveolar carcinoma − NCI-H358 lungbronchioalveolar carcinoma, from − metastatic site alveolus NCI-H441lung papillary adenocarcinoma ++ NCI-H460 lung Large cell lungcarcinoma, from − metastatic site: pleural effusion NCI-H520 lungsquamous cell carcinoma − NCI-H522 lung adenocarcinoma K-ras, mutated −p53 NCI-H661 lung large cell lung carcinoma, from − metastatic site:lymph node NCI-H69 (SCLC) lung − NCI-H82 (SCLC) lung from metastic sit:epleural effusion − SW 900 lung squamous cell carcinoma − HCC-2935 lungadenocarcinoma ++++ HCC-1395 lung primary ductal carcinoma − RPMI-8226myeloma plasmacytoma; multiple myeloma + (IgG lambda-type) B lymphocyte;lymphoblast SKMM2 myeloma − L-363 myeloma plasma cell leukemia − JJN-3myeloma plasma cell leukemia − KMS-12-BM myeloma − KMS-12-PE myeloma −LP-1 myeloma − MOLP-2 myeloma MOLP-8 myeloma − SNU-1 gastric frommetastatic site: ascites +++ Hela cervix adenocarcinoma − DMS-153 (SCLC)lung from metastatic site: liver − MeWo skin fibroblast, malignantmelanoma, − from metastic site: lymph node Definition of −, +, ++, +++as determined from FACS median fluorescence log shift: − = no shift; + =log shift as compared to control antibody, median 10-100; ++ = 2 logshift, median 100-1,000; +++ = 3 log shift, median 1,000-10,000; ++++ =4 log shit median>10000 *All cell lines from public tissue bank such asthe American tissue culture collection etc, except KS breast cancer cellline which was kindly provided by Dr. Gückel (Tübingen); B. Gueckel,Cancer Cell International 2004, 4(Suppl 1): S38).

In conclusion, CEACAM6 is expressed in cell lines which representvarious cancers (e.g. colorectal cancer, non-small-cell lung cancer(NSCLC), small cell lung cancer (SCLC), pancreatic cancer, gastriccancer, breast cancer and multiple myeloma) which constitute potentialtarget indications for treatment with CEACAM6 immunomodulatingantibodies and other response modifiers (e.g. peptides, small molecules,artificial scaffold binders etc).

Example 15 Binding to Single Domain 1 of Human and Cynomolgus CEACAM6

To test whether antibodies of the invention can bind to the isolatedsingle domain 1 of human and cynomolgus CEACAM6, SPR experiments wereconducted as in Example 1.

The single N-terminal domain 1 of cynomolgus CEACAM6 (TPP-2453) wasproduced as described in Example 1 in analogy to TPP-1794:

Name Protein-ID Description SEQ-ID Macaca fascicularis TPP-2453Domain 1, fusion to SEQ-ID NO: 180 CEACAM6-Domain 1-His (expressed in E. His coli) SEQ-ID NO: 180 (TPP-2453)MQLTIESRPFNVAEGKEVLLLAHNLPQNTLGFNWYKGERVDAKRLIVAYVIGTQQTTPGPAHSGREMIYSITASLLIQNVTQNDTGSYTLQAIKEDLVTEEATGRFWVYPELGSGSHHHHHHHHAffinities (monovalent K_(D)) of antibodies of the invention towardsrecombinant single domain 1 of human and cynomolgus CEACAM6 weredetermined by SPR analogously to experimental procedures described inExample 1, and are shown in Table 27.

TABLE 27 SPR analysis: monovalent K_(D) (in nM) Recombinant Recombinantsingle single domain 1 domain 1 of Macaca of human fascicularis CEACAM6CEACAM6 Test (TPP-1794) (TPP-2453) Antibody Isotype K_(D) [nM] K_(D)[nM] TPP-2971 Mouse IgG1 11 5.3 TPP-3186 Mouse IgG1 15 8.7 TPP-3187Mouse IgG1 12 6.9 TPP-3308 Human IgG2 9.5 5.4 chimera TPP-3310 HumanIgG2 3.7 3.4 TPP-3322 Human IgG2 12 8.8 chimera TPP-3323 Human IgG2 96.9 chimera TPP-3705 Human IgG2 6.7 2.7 TPP-3707 Human IgG2 6.4 2.4TPP-3714 Human IgG2 3.7 3.2 TPP-3820 Human IgG2 5.1 5.2 TPP-3821 HumanIgG2 4.5 4.7 TPP-1745 Human IgG1 3.3 − “−”: no binding detectedIn conclusion, antibodies of the invention bind to both human andcynomolgus N-terminal domain 1 of CEACAM6 with comparable affinities.

Example 16 X-Ray Crystal Structure of the Human CEACAM6 SingleN-Terminal Domain1 in Complex with Fab Fragment APP-1574

The crystal structure of single N-terminal domain 1 of human CEACAM6(TPP-1794; SEQ ID NO 169) bound to a Fab fragment related to TPP-3310(called APP-1574) was determined.

To facilitate production of the Fab fragment TPP-3310 was produced ashuman IgG1 variant (called TPP-5468, see Table 28). Papain cleavage ofTPP-5468 and subsequent purification results in APP-1574. This Fabfragment comprises the variable domains (VH and VL) of TPP-3310 (seeTable 28).

TABLE 28 Amino acid sequences of IgG and Fab used for crystal structuredetermination Protein Heavy Chain Light Chain TPP-5468 SEQ ID NO: 181SEQ ID NO: 182 APP-1574 SEQ ID NO: 183 SEQ ID NO: 184

As detailed in Example 1, CEACAM6 domain 1 has been expressed andrefolded from E. coli. The Fab-fragment has been generated by digestionof the antibody with Papain, followed by complex formation. Proteincrystallography was then employed to generate atomic resolution forsingle N-terminal domain 1 of human CEACAM6 bound to APP-1574 Fab todefine the epitope.

Protein Production

The single N-terminal domain 1 of human CEACAM6 (TPP-1794; SEQ-ID 169)was produced as 6× His fused protein construct as described inExample 1. The protein was concentrated to 6.7 mg/ml prior to complexformation.

The corresponding Fab-fragment of TPP-5468 (human IgG1) has beenobtained by cleavage with the protease Papain. 1 mg of the antibody wasmixed with 50 pl immobilized papain (ThermoFisher #20341) in digestionbuffer (20 mM Na-Phosphate pH 7.0, 10 mM EDTA, 20 mM Cystein-HCl) andincubated for 4 h at 37° C. with continuous stirring. Immobilized papainwas removed by centrifugation and resulting Fc-fragments and non-cleavedIgG were removed by passing over MabSelectSURE (GE Healthcare,#11-0034-89 AC). The Fab-fragment in the flow-through was furtherpurified via size exclusion chromatography in 30 mM Tris buffer pH 8.5,150 mM NaCl on Superdex 75 and concentrated to 7.2 mg/ml.

For complex formation, purified Fab-fragment and human CEACAM6N-terminal domain 1 were mixed in ratio 1 Fab to 1.4 human CEACAM6N-terminal domain 1 for 1 human CEACAM6 N-terminal domain 1 h at 4° C.The resulting protein complex was isolated by size exclusionchromatography in 30 mM Tris buffer pH 8.5, 150 mM NaCl on Superdex 75and further concentrated to 21.2 mg/ml prior to crystallization.

Crystallization and Structure Determination

The complex of single N-terminal domain1 of human CEACAM6 and the Fabfragment APP-1574 was concentrated to 21.2 mg/ml, centrifuged at 20,000g for 10 minutes and screened for crystallization. Crystals for datacollection were grown by hanging drop vapor diffusion at 20° C. Indetail, 0.2 μl of the complex was mixed with 0.2 μl of reservoirsolution containing 100 mM tri-sodium citrate pH 4.9, 19% (w/v) PEG 4000and 10% (v/v) isopropanol. The drop was then equilibrated against 80 μlof the same reservoir solution. Before data collection, the crystalswere flash cooled in liquid nitrogen.

Diffraction data were collected at beamline 14-1 at the BESSY IISynchrotron Source (Helmholtz Zentrum Berlin) and processed using XDS(Kabsch, W. XDS. Acta Cryst. D66, 125-132 (2010). The data of humanCEACAM6 single N-terminal domain1-Fab fragment APP-1574 complex wereprocessed to 2.7 A in the space group P1 with cell dimensions a=64.7 Å,b=65.2 Å, c=78.6 Å, alpha=66.1°, beta=87.2° and gamma=88.5°. Thestructure of the complex was solved by molecular replacement usingPHASER (McCoy AJ et al, J Appl Cryst (2007). 40, 658-674). with in-housestructures of the human CEACAM6 single N-terminal domain1 and a Fab assearch models. The final model was built in COOT (Emsley, P. et al, ActaCryst D66, 486-501 (2010)) and refined using CCP4 (Winn, M. D. et al.Acta. Cryst. D67, 235-242 (2011)).

The epitope was defined as residues of human CEACAM6 single N-terminaldomain1 that contain atoms within 5 Å to any atom in Fab fragmentAPP-1574, identified by NCONT in the CCP4 program suite (Winn, M. D. etal. Acta. Cryst. D67, 235-242 (2011)) and listed in Table 29). There aretwo copies of human CEACAM6 single N-terminal domain1-Fab fragmentAPP-1574 complex in the asymmetric unit (the smallest unique unit in thecrystal). Only those antibody-contacting residues that are common inboth copies are listed as epitope residues.

Epitope

The crystal structure of the human CEACAM6 single N-terminal domain1-Fabfragment APP-1574 complex was used to identify the epitope of Fabfragment APP-1574 on CEACAM6. The interaction surface on human CEACAM6single N-terminal domain1 by Fab fragment APP-1574 is formed by severalcontinuous and discontinuous (i.e. noncontiguous) sequences; namelyresidues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Va183, Ile84, Gly85,Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 (numberingaccording to SEQ-ID:179; TPP-4639) as detailed in Table 29.

In a very close direct contact are residues having at least one atomthat is 3.6 Å or less away from the antibody. These residues are: Gln60,Asn61, Arg62, Ile63, Va183, Ile84, Gly85, Thr90, Ser127, Asp128 andLeu129 (numbering according to SEQ-ID:179; TPP-4639).

These residues form the exemplary three-dimensional conformationalepitope that is recognized by the Fab fragment APP-1574 (FIGS. 9 and10).

TABLE 29 Interactions between single N-terminal domain 1 of humanCEACAM6 and Fab APP-1574 N-terminal domain 1 of human CEACAM6 (TPP-1794;SEQ-ID 169) Number as in SEQ-ID 169 (number as in SEQ-ID NO: 179 FabAPP-1574 Amino Acid in brackets) Amino Acid Number Chain Pro 26 (59) Tyr32 H Gln 27 (60) Tyr 32 H Asn 28 (61) Trp 55 H Asn 56 H Tyr 32 H Asn 58H Arg 29 (62) Trp 55 H Tyr 32 H Gly 33 H Ile 30 (63) Ser 101 H Gly 33 HTrp 54 H Leu 102 H Ser 101 H Pro 103 H Trp 55 H Gly 31 (64) Leu 102 HVal 50 (83) Ser 101 H Leu 102 H Tyr 104 H Ile 51 (84) Gly 33 H Tyr 32 HGly 52 (85) Ile 34 H Gly 33 H Ser 101 H Tyr 32 H Thr 31 H Thr 53 (86)Ser 101 H Ile 34 H Arg 99 H Gln 55 (88) Tyr 49 L Thr 57 (90) Tyr 49 LAsn 53 L Pro 58 (91) Asn 53 L Ile  92 (125) Leu 102 H Ser  94 (127) Tyr94 L Trp 54 H Tyr 60 H Trp 55 H Asn 56 H Asn 58 H Asp  95 (128) Tyr 94 LTyr 60 H Leu  96 (129) Tyr 94 L Ser 92 L Ser 93 L Tyr 91 L Pro 103 H

CEACAM6 N-terminal domain1 residues numbered as in SEQ ID NO 169. Theantibody residues are numbered based upon their linear amino acidsequence (SEQ ID NO: 183 and SEQ ID NO: 184) and corresponding chainsare labeled (“H” for heavy chain, “L” for light chain). Human CEACAM6single N-terminal domain1 residues shown here to have at least one atomwith 5 Å to any atom in Fab fragment APP-1574, to account for potentialwater mediated interactions.

When carefully analyzing the epitope, it becomes apparent thatIsoleucine-63 (according to SEQ-ID NO: 179) of human CEACAM6 is acentral part of the epitope. The Isoleucine side-chain has good shapecomplementary with APP-1574. Modelling indicates that a Leucine at thisposition in cynomolgus CEACAM6 can be sterically accommodated and willnot disrupt the interaction. This explains a retained binding activityto cynomolgus CEACAM6 activity and is the basis for human-cynomolguscross-reactivity. In contrast, a Phenylalanine at this position (as inhuman CEACAM1, human CEACAM3 and human CEACAM5) cannot be stericallyaccommodated and will lead to loss in binding activity. This is thebasis for CEACAM6 selectivity.

Thereby, recognition of Isoleucine-63 (according to SEQ-ID NO: 179)represents the most significant “selectivity tuner” between targetCEACAMs. The APP-1574 recognition mode of human CEACAM6 optimallyexploits key residue difference between targets and off-targets.Previous analysis of a structure of the Fab fragment of TPP-1679 (whichselectivity profile is insufficient, see Example 4) in complex withN-terminal domain 1 also identified Isoleucine-63 (according to SEQ-IDNO: 179) as potential selectivity switch (data not shown). However,since the molecular recognition mechanism by TPP-1679 is different,whereby Ileucine-63 (according to SEQ-ID NO: 179) is located at thebinding site periphery, it was difficult to exploit.

In summary, binders that in addition to other residues forming theepitope (Table 29) also optimally exploit binding to the selectivity &cross-reactivity determining residue Isoleucine-63 (according to SEQ-ID0: 179) of CEACAM6 and still allowing the accommodation of a Leucine atthat position, but not Phenylalanine, will be CEACAM6 selective, but atthe same time human-cynomolgus CEACAM6 cross-reactive.

Mutagenesis

To substantiate the findings and predictions of structural analysis inbinding studies, the following mutants of N-terminal domain 1 of humanCEACAM6 were generated:

TABLE 30Amino acid sequences of proteins used in mutational binding studiesProtein Protein-ID Amino Acid Sequence Human CEACAM6- TPP-1794;MKLTIESTPF NVAEGKEVLL LAHNLPQNR I Domain 1-His SEQ-IDGYSWYKGERV DGNSLIVGYV IGTQQATPGP Wild-type NO: 169 AYSGRETIYP NASLLIQNVTQNDTGFYTLQ VIKSDLVNEE ATGQFHVYPG SGSHHHHHHH H Human CEACAM6- TPP-8697MKLTIESTPF NVAEGKEVLL LAHNLPQNR L Domain 1-His- SEQ-IDGYSWYKGERV DGNSLIVGYV IGTQQATPGP 130L (I63L according NO: 185AYSGRETIYP NASLLIQNVT to SEQ-ID NO: 179)QNDTGFYTLQ VIKSDLVNEE ATGQFHVYPG SGSHHHHHHH H Human CEACAM6- TPP-8698MKLTIESTPF NVAEGKEVLL LAHNLPQNR F Domain 1-His- SEQ-IDGYSWYKGERV DGNSLIVGYV IGTQQATPGP 130F (I63F according NO: 186AYSGRETIYP NASLLIQNVT to SEQ-ID NO: 179)QNDTGFYTLQ VIKSDLVNEE ATGQFHVYPG SGSHHHHHHH HThe proteins were expressed in E. coli, refolded and purified asdescribed in Example 1. Comparative binding activity to domain 1wild-type protein and the two single mutations was determined by ELISAmethod. 1.5 ug/ml protein solutions in PBS were coated overnight to 384Nunc MaxiSorp plates (Sigma, P6491). Plates were washed with PBS/T andblocked with Smart block (CANDOR Bioscience GmbH, 113125). Subsequently,dilution series of TPP-3310, TPP-1679 and an isotype control antibodywere applied to the wells. After washing with PBS/T, bound antibodieswere detected with Anti Human IgG Fc POD (Sigma, A0170) and 10 μM AmplexRed solution (Thermo, A12222). Positive binding signals were detectedvia Fluorescence (Ex. 535 nm/Em. 590 nm). Table 32 shows bindingactivity for TPP-3310 to the domain 1 wild-type protein and the mutationof Isoleucine 63 (as in SEQ-ID NO: 179) to Leucine (as in cynomolgus).The mutation of amino acid position 63 (as in SEQ-ID NO: 179) toPhenylalanine (as in human CEACAM1, human CEACAM3 and human CEACAM5)results in complete loss of binding ability. As a control fordemonstration of efficient refolding of CEACAM6 N-terminal domain 1proteins, TPP-1679 was employed (see Example 4), for which binding to asimilar extent to all antigens tested in Table 31 was observed.

TABLE 31 Binding activity on CEACAM6 N-terminal domain 1 mutants. HumanHuman CEACAM6- CEACAM6- Domain Domain Human 1-His 1-His CEACAM6- I30L(I63L I30F (I63F Domain according according 1-His to SEQ-ID to SEQ-IDWild-type NO: 179) NO: 179) TPP-1794; TPP-8697; TPP-8698; SEQ-ID SEQ-IDSEQ-ID Antibody NO: 169 NO: 185 NO: 186 TPP-3310 + + − TPP-1679 + + +Isotype control − − − “+” denotes binding detected; “−” denotes nobinding detected

In conclusion, the truly human—cynomolgus CEACAM6 cross-reactiveantibody TPP-3310, which is at the same time selective with regards toother human paralogs, was able to tolerate an 163L substitution(according to SEQ-ID: 179) (corresponding to cynomolgus CEACAM6 residue)but not an 163F substitution (according to SEQ-ID: 179) (correspondingresidue in human paralogs CEACAM1, CEACAM3, and CEACAM5) in the contextof human CEACAM6 N-terminal domain 1 consistent with results obtainedfrom X-ray crystallography and substantiating our prediction.

Example 17 Analysis of T Cell Mediated Cytotoxicity in the Presence ofCEACAM6 Antibodies

The effect of the anti-CEACAM6 antibodies on T cell mediatedcytotoxicity was studied in cytotoxicity experiments with co-cultures ofCEACAM6 positive tumor cells and T cells derived of different sources.These T cells were either CD8⁺ survivin T cells or patient-derived Tcells from a pancreatic cancer. For these tumor cell killing experimentsan impedance based cytotoxicity assay (xCELLigence) system was used.

The survivin-peptide specific CD8⁺ T cell clone was generated andexpanded in vitro as described in Example 11. Pancreatic cancer tumorinfiltrating lymphocyte cell lines (TILs) were isolated from freshprimary culture of tumor tissue from surgery. In brief, fresh primarytissue material was cut into small pieces and cultured in small dishesin X-Vivo-15 medium (Lonza) containing 2% human serum albumin, 2.5 μg/mlFungizone, 20 μg/ml Gentamycin, 1% Penicillin/Streptomycin with 6000IU/IL-2 for 10-18 days. Afterwards cells from the supernatant wereharvested and either frozen or used directly for a “rapid expansionprotocol” (REP). For rapid expansion of TILs, frozen TILs were gentlythawed and cultured with 0.6*10⁶ cells/ml for 1 day in CompleteLymphocyte Medium CLM RPMI-1640 (Life Technologies #21875034), 10% humanAB Serum (MILAN Analytica #000083), 1% Penicillin/Streptomycin (LifeTechnologies #15140122), 1% ml HEPES (Life Technolgies #15630056), 0.01%β-mercaptoethanol [stock 50 mM] (Life Technologies #31350010)) with 6000IU/ml IL-2. TILs were harvested and expanded at a 1:100 ratio with 60 Gyirradiated feeder PBMCs from 3 different donors in 400 ml REP medium(50% CLM mixed with 50% AIM-V serum free medium (Gibco #12055091)containing 3000 IU/ml IL-2 and 30 ng/ml OKT-3 antibody (eBioscience#16-0037-85)) in G-REX-100 Flasks (Wilson Wolf #80500S). Cells werecultured and splitted as described in Jin et al., J Immunother. 2012April; 35(3):283-92 . After 14 days cells were harvested and frozen inaliquots. Prior to co-culture cytotoxicity assays, individual aliquotsof TILs were gently thawed and cultured with 0.6*10⁶ cells/ml for 2 daysin CLM containing 6000 IU/ml IL-2 and 1 day in CLM without IL-2.

Tumor Cells were Cultivated According to Standard Protocols andProvider's Instructions

T cell mediated cytotoxity was analyzed in an impedance basedcytotoxicity assay (xCELLigence) system. In this label free assay systemcytotoxicity is measured directly and continuously over a long timeperiod of around 100-150 h (real time). Adherent tumor cells areattached to microelectrodes at the bottom of a 96-Well E-plate (E-PlateVIEW 96 PET; ACEA Biosciences #ID:H000568) which changes the electricalimpedance of these electrodes. This is monitored as an increase of thedimensionless “cell index”. After adherence of the tumor cells (˜24 h)antibodies and T cells are added to the wells which, if T cells exertcytotoxic activity, results in lysis of the tumor cells and detachmentfrom the electrodes. This detachment changes the impedance of the wellsand is measured as a decrease of the “cell index” or “normalized cellindex” which is the “cell index” normalized to the time point of T celladdition. The T cells alone do not affect the electrical impedance ofthe electrodes and thus only the cytolysis of the tumor cells ismeasured. (Peper et al, J Immunol Methods. 2014 March; 405:192-8)

In first experiments we established that tumor cell killing observed inthis assay system is T cell dose dependent and works for different tumorcell:T cell ratios and different T cell sources (Survivin-peptidespecific CD8⁺ T cells, TILs from pancreatic cancer patients).

We then studied the effect of anti-CEACAM6 antibodies on the cytolyticefficacy of survivin T cells. Therefore, the CEACAM6 positive breastcancer KS or the CEACAM6 transfected colon cancer HCT-116 (HCT116-hC6)was added to 96-well plates for 24 h before survivin-peptide specific Tcells were added at different cell ratios together with the anti-CEACAM6mAbs. The coculture was followed for a time period ˜100 h. In theseexperiments we observed an improved T cell dependent cytotoxicity in thepresence of the anti-CEACAM6 antibodies TPP-3310 and TPP-3470 of ˜21% onboth cell lines. The results are displayed in FIGS. 11A and B)exemplarily for one cell ratio. Notably, the survivin-peptide specificCD8⁺ T cells alone show already a high cytotoxic impact of 45-62%, whichis most likely due to the preactivation of the cultured T cells and isthus considered as background cytolysis. In summary, the increase ofIFN-gamma secretion observed in the previous ELISA assays translatesinto a cytotoxic effect within approximately 24 h of co-culture. Weconclude that treatment of CEACAM6 positive tumor cells withanti-CEACAM6 antibodies leads to improved survivin-peptide specific CD8⁺T cell mediated killing of both tumor cell lines.

In subsequent experiments we tested the effect of the CEACAM6 antibodieson the cytolytic activity of patient-derived TILs cells of a pancreaticcancer. Therefore, the CEACAM6 positive lung cancer cell line HCC2935was added to 96-well plates and cultivated for 24 h. Then, TILs wereadded at different ratios in the presence of the CEACAM6 antibody (30pg/ml) and of a bispecific antibody anti-CD3×anti-EPCAM IgG (0.25 ng/ml)(Marme et al., Int J Cancer. 2002 Sep. 10; 101(2)183-9; Salnikov et al.,J Cell Mol Med. 2009 September; 13(9B):4023-33) to allow forHLA-independent T cell mediated tumor cell killing. In the presence ofthe anti-CEACAM6 antibodies TPP-3310 and TPP-3470 we observed a completedrop of impedance which was not observed in the presence of the isotypematched control antibody. The drop in impedance is interpreted ascomplete cytolytic kill of the target cell line HCC2935. In anadditional experiment it could be demonstrated that the effect of theCEACAM6 antibody TPP-3310 is dose dependent and an IC₅₀ value of0.62-0.21 μg/ml was determined. FIG. 12 shows exemplarily the resultsfor TIL-12.

In summary these experiments show that the CEACAM6 antibodies of theinvention have the potential to effectively block the immunosuppressivereceptor CEACAM6 and improve the cytotoxic efficacy not only of model Tcells but also of patient-derived Tumor infiltrating lymphocytes againstCEACAM6 positive tumor cells.

Table of Sequences

TABLE 32 Correlation of SEQ ID NO to TPP-ID and associated sequencefeatures (heavy and light chain of antibody, variable regions,complementarity determining regions (CDR)) for proteins (PRT) andnucleic acids (DNA) “Sequence “TPP ID” “Sequence Name” Region” “SequenceType” “SEQ ID” TPP-1173 h16C3-hIgG1 Heavy Chain PRT SEQ ID NO: 1TPP-1173 h16C3-hIgG1 Light Chain PRT SEQ ID NO: 2 TPP-2971 792.15H12C9VH PRT SEQ ID NO: 3 TPP-2971 792.15H12C9 HCDR1 PRT SEQ ID NO: 4 TPP-2971792.15H12C9 HCDR2 PRT SEQ ID NO: 5 TPP-2971 792.15H12C9 HCDR3 PRT SEQ IDNO: 6 TPP-2971 792.15H12C9 VL PRT SEQ ID NO: 7 TPP-2971 792.15H12C9LCDR1 PRT SEQ ID NO: 8 TPP-2971 792.15H12C9 LCDR2 PRT SEQ ID NO: 9TPP-2971 792.15H12C9 LCDR3 PRT SEQ ID NO: 10 TPP-3186 792.11G2D10 VH PRTSEQ ID NO: 13 TPP-3186 792.11G2D10 HCDR1 PRT SEQ ID NO: 14 TPP-3186792.11G2D10 HCDR2 PRT SEQ ID NO: 15 TPP-3186 792.11G2D10 HCDR3 PRT SEQID NO: 16 TPP-3186 792.11G2D10 VL PRT SEQ ID NO: 17 TPP-3186 792.11G2D10LCDR1 PRT SEQ ID NO: 18 TPP-3186 792.11G2D10 LCDR2 PRT SEQ ID NO: 19TPP-3186 792.11G2D10 LCDR3 PRT SEQ ID NO: 20 TPP-3187 792.15C4F4 VH PRTSEQ ID NO: 23 TPP-3187 792.15C4F4 HCDR1 PRT SEQ ID NO: 24 TPP-3187792.15C4F4 HCDR2 PRT SEQ ID NO: 25 TPP-3187 792.15C4F4 HCDR3 PRT SEQ IDNO: 26 TPP-3187 792.15C4F4 VL PRT SEQ ID NO: 27 TPP-3187 792.15C4F4LCDR1 PRT SEQ ID NO: 28 TPP-3187 792.15C4F4 LCDR2 PRT SEQ ID NO: 29TPP-3187 792.15C4F4 LCDR3 PRT SEQ ID NO: 30 TPP-3308 TPP-2971X1- VH PRTSEQ ID NO: 33 hIgG2Kappa TPP-3308 TPP-2971X1- HCDR1 PRT SEQ ID NO: 34hIgG2Kappa TPP-3308 TPP-2971X1- HCDR2 PRT SEQ ID NO: 35 hIgG2KappaTPP-3308 TPP-2971X1- HCDR3 PRT SEQ ID NO: 36 hIgG2Kappa TPP-3308TPP-2971X1- VL PRT SEQ ID NO: 37 hIgG2Kappa TPP-3308 TPP-2971X1- LCDR1PRT SEQ ID NO: 38 hIgG2Kappa TPP-3308 TPP-2971X1- LCDR2 PRT SEQ ID NO:39 hIgG2Kappa TPP-3308 TPP-2971X1- LCDR3 PRT SEQ ID NO: 40 hIgG2KappaTPP-3308 TPP-2971X1- VH DNA SEQ ID NO: 41 hIgG2Kappa TPP-3308TPP-2971X1- VL DNA SEQ ID NO: 42 hIgG2Kappa TPP-3308 TPP-2971X1- HeavyChain PRT SEQ ID NO: 43 hIgG2Kappa TPP-3308 TPP-2971X1- Light Chain PRTSEQ ID NO: 44 hIgG2Kappa TPP-3308 TPP-2971X1- Heavy Chain DNA SEQ ID NO:45 hIgG2Kappa TPP-3308 TPP-2971X1- Light Chain DNA SEQ ID NO: 46hIgG2Kappa TPP-3310 TPP-2971HU1- VH PRT SEQ ID NO: 47 hIgG2KappaTPP-3310 TPP-2971HU1- HCDR1 PRT SEQ ID NO: 48 hIgG2Kappa TPP-3310TPP-2971HU1- HCDR2 PRT SEQ ID NO: 49 hIgG2Kappa TPP-3310 TPP-2971HU1-HCDR3 PRT SEQ ID NO: 50 hIgG2Kappa TPP-3310 TPP-2971HU1- VL PRT SEQ IDNO: 51 hIgG2Kappa TPP-3310 TPP-2971HU1- LCDR1 PRT SEQ ID NO: 52hIgG2Kappa TPP-3310 TPP-2971HU1- LCDR2 PRT SEQ ID NO: 53 hIgG2KappaTPP-3310 TPP-2971HU1- LCDR3 PRT SEQ ID NO: 54 hIgG2Kappa TPP-3310TPP-2971HU1- VH DNA SEQ ID NO: 55 hIgG2Kappa TPP-3310 TPP-2971HU1- VLDNA SEQ ID NO: 56 hIgG2Kappa TPP-3310 TPP-2971HU1- Heavy Chain PRT SEQID NO: 57 hIgG2Kappa TPP-3310 TPP-2971HU1- Light Chain PRT SEQ ID NO: 58hIgG2Kappa TPP-3310 TPP-2971HU1- Heavy Chain DNA SEQ ID NO: 59hIgG2Kappa TPP-3310 TPP-2971HU1- Light Chain DNA SEQ ID NO: 60hIgG2Kappa TPP-3322 TPP-3186X1-hIgG2 VH PRT SEQ ID NO: 61 TPP-3322TPP-3186X1-hIgG2 HCDR1 PRT SEQ ID NO: 62 TPP-3322 TPP-3186X1-hIgG2 HCDR2PRT SEQ ID NO: 63 TPP-3322 TPP-3186X1-hIgG2 HCDR3 PRT SEQ ID NO: 64TPP-3322 TPP-3186X1-hIgG2 VL PRT SEQ ID NO: 65 TPP-3322 TPP-3186X1-hIgG2LCDR1 PRT SEQ ID NO: 66 TPP-3322 TPP-3186X1-hIgG2 LCDR2 PRT SEQ ID NO:67 TPP-3322 TPP-3186X1-hIgG2 LCDR3 PRT SEQ ID NO: 68 TPP-3322TPP-3186X1-hIgG2 VH DNA SEQ ID NO: 69 TPP-3322 TPP-3186X1-hIgG2 VL DNASEQ ID NO: 70 TPP-3322 TPP-3186X1-hIgG2 Heavy Chain PRT SEQ ID NO: 71TPP-3322 TPP-3186X1-hIgG2 Light Chain PRT SEQ ID NO: 72 TPP-3322TPP-3186X1-hIgG2 Heavy Chain DNA SEQ ID NO: 73 TPP-3322 TPP-3186X1-hIgG2Light Chain DNA SEQ ID NO: 74 TPP-3323 TPP-3187X1-hIgG2 VH PRT SEQ IDNO: 75 TPP-3323 TPP-3187X1-hIgG2 HCDR1 PRT SEQ ID NO: 76 TPP-3323TPP-3187X1-hIgG2 HCDR2 PRT SEQ ID NO: 77 TPP-3323 TPP-3187X1-hIgG2 HCDR3PRT SEQ ID NO: 78 TPP-3323 TPP-3187X1-hIgG2 VL PRT SEQ ID NO: 79TPP-3323 TPP-3187X1-hIgG2 LCDR1 PRT SEQ ID NO: 80 TPP-3323TPP-3187X1-hIgG2 LCDR2 PRT SEQ ID NO: 81 TPP-3323 TPP-3187X1-hIgG2 LCDR3PRT SEQ ID NO: 82 TPP-3323 TPP-3187X1-hIgG2 VH DNA SEQ ID NO: 83TPP-3323 TPP-3187X1-hIgG2 VL DNA SEQ ID NO: 84 TPP-3323 TPP-3187X1-hIgG2Heavy Chain PRT SEQ ID NO: 85 TPP-3323 TPP-3187X1-hIgG2 Light Chain PRTSEQ ID NO: 86 TPP-3323 TPP-3187X1-hIgG2 Heavy Chain DNA SEQ ID NO: 87TPP-3323 TPP-3187X1-hIgG2 Light Chain DNA SEQ ID NO: 88 TPP-3688h16C3-hIgG2Kappa Heavy Chain PRT SEQ ID NO: 89 TPP-3688 h16C3-hIgG2KappaLight Chain PRT SEQ ID NO: 90 TPP-3705 090E-M007-A09- VH PRT SEQ ID NO:91 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- HCDR1 PRT SEQ ID NO:92 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- HCDR2 PRT SEQ ID NO:93 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- HCDR3 PRT SEQ ID NO:94 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- VL PRT SEQ ID NO: 95Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- LCDR1 PRT SEQ ID NO: 96Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- LCDR2 PRT SEQ ID NO: 97Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- LCDR3 PRT SEQ ID NO: 98Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- VH DNA SEQ ID NO: 99Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- VL DNA SEQ ID NO: 100Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- Heavy Chain PRT SEQ IDNO: 101 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- Light Chain PRTSEQ ID NO: 102 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09- HeavyChain DNA SEQ ID NO: 103 Mat1-hIgG2- hIgG2Kappa TPP-3705 090E-M007-A09-Light Chain DNA SEQ ID NO: 104 Mat1-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- VH PRT SEQ ID NO: 105 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- HCDR1 PRT SEQ ID NO: 106 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- HCDR2 PRT SEQ ID NO: 107 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- HCDR3 PRT SEQ ID NO: 108 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- VL PRT SEQ ID NO: 109 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- LCDR1 PRT SEQ ID NO: 110 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- LCDR2 PRT SEQ ID NO: 111 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- LCDR3 PRT SEQ ID NO: 112 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- VH DNA SEQ ID NO: 113 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- VL DNA SEQ ID NO: 114 Mat2-hIgG2- hIgG2Kappa TPP-3707090E-M007-A09- Heavy Chain PRT SEQ ID NO: 115 Mat2-hIgG2- hIgG2KappaTPP-3707 090E-M007-A09- Light Chain PRT SEQ ID NO: 116 Mat2-hIgG2-hIgG2Kappa TPP-3707 090E-M007-A09- Heavy Chain DNA SEQ ID NO: 117Mat2-hIgG2- hIgG2Kappa TPP-3707 090E-M007-A09- Light Chain DNA SEQ IDNO: 118 Mat2-hIgG2- hIgG2Kappa TPP-3714 TPP-2971HU2- VH PRT SEQ ID NO:119 hIgG2Kappa TPP-3714 TPP-2971HU2- HCDR1 PRT SEQ ID NO: 120 hIgG2KappaTPP-3714 TPP-2971HU2- HCDR2 PRT SEQ ID NO: 121 hIgG2Kappa TPP-3714TPP-2971HU2- HCDR3 PRT SEQ ID NO: 122 hIgG2Kappa TPP-3714 TPP-2971HU2-VL PRT SEQ ID NO: 123 hIgG2Kappa TPP-3714 TPP-2971HU2- LCDR1 PRT SEQ IDNO: 124 hIgG2Kappa TPP-3714 TPP-2971HU2- LCDR2 PRT SEQ ID NO: 125hIgG2Kappa TPP-3714 TPP-2971HU2- LCDR3 PRT SEQ ID NO: 126 hIgG2KappaTPP-3714 TPP-2971HU2- VH DNA SEQ ID NO: 127 hIgG2Kappa TPP-3714TPP-2971HU2- VL DNA SEQ ID NO: 128 hIgG2Kappa TPP-3714 TPP-2971HU2-Heavy Chain PRT SEQ ID NO: 129 hIgG2Kappa TPP-3714 TPP-2971HU2- LightChain PRT SEQ ID NO: 130 hIgG2Kappa TPP-3714 TPP-2971HU2- Heavy ChainDNA SEQ ID NO: 131 hIgG2Kappa TPP-3714 TPP-2971HU2- Light Chain DNA SEQID NO: 132 hIgG2Kappa TPP-3820 3187HU1- VH PRT SEQ ID NO: 133 hIgG2KappaTPP-3820 3187HU1- HCDR1 PRT SEQ ID NO: 134 hIgG2Kappa TPP-3820 3187HU1-HCDR2 PRT SEQ ID NO: 135 hIgG2Kappa TPP-3820 3187HU1- HCDR3 PRT SEQ IDNO: 136 hIgG2Kappa TPP-3820 3187HU1- VL PRT SEQ ID NO: 137 hIgG2KappaTPP-3820 3187HU1- LCDR1 PRT SEQ ID NO: 138 hIgG2Kappa TPP-3820 3187HU1-LCDR2 PRT SEQ ID NO: 139 hIgG2Kappa TPP-3820 3187HU1- LCDR3 PRT SEQ IDNO: 140 hIgG2Kappa TPP-3820 3187HU1- VH DNA SEQ ID NO: 141 hIgG2KappaTPP-3820 3187HU1- VL DNA SEQ ID NO: 142 hIgG2Kappa TPP-3820 3187HU1-Heavy Chain PRT SEQ ID NO: 143 hIgG2Kappa TPP-3820 3187HU1- Light ChainPRT SEQ ID NO: 144 hIgG2Kappa TPP-3820 3187HU1- Heavy Chain DNA SEQ IDNO: 145 hIgG2Kappa TPP-3820 3187HU1- Light Chain DNA SEQ ID NO: 146hIgG2Kappa TPP-3821 3187HU2- VH PRT SEQ ID NO: 147 hIgG2Kappa TPP-38213187HU2- HCDR1 PRT SEQ ID NO: 148 hIgG2Kappa TPP-3821 3187HU2- HCDR2 PRTSEQ ID NO: 149 hIgG2Kappa TPP-3821 3187HU2- HCDR3 PRT SEQ ID NO: 150hIgG2Kappa TPP-3821 3187HU2- VL PRT SEQ ID NO: 151 hIgG2Kappa TPP-38213187HU2- LCDR1 PRT SEQ ID NO: 152 hIgG2Kappa TPP-3821 3187HU2- LCDR2 PRTSEQ ID NO: 153 hIgG2Kappa TPP-3821 3187HU2- LCDR3 PRT SEQ ID NO: 154hIgG2Kappa TPP-3821 3187HU2- VH DNA SEQ ID NO: 155 hIgG2Kappa TPP-38213187HU2- VL DNA SEQ ID NO: 156 hIgG2Kappa TPP-3821 3187HU2- Heavy ChainPRT SEQ ID NO: 157 hIgG2Kappa TPP-3821 3187HU2- Light Chain PRT SEQ IDNO: 158 hIgG2Kappa TPP-3821 3187HU2- Heavy Chain DNA SEQ ID NO: 159hIgG2Kappa TPP-3821 3187HU2- Light Chain DNA SEQ ID NO: 160 hIgG2KappaTPP-1306 macaca mulatta Chain 1 PRT SEQ ID NO: 161 CEACAM6-Xa-Fc- HisTPP-1436 Ceacam6 Chain 1 PRT SEQ ID NO: 162 TPP-1437 Ceacam1 Chain 1 PRTSEQ ID NO: 163 TPP-1438 Ceacam5 Chain 1 PRT SEQ ID NO: 164 TPP-1790hCeacam6-WT-Fc- Chain 1 PRT SEQ ID NO: 165 6xHis TPP-1791 hCeacam6-Dom1-Chain 1 PRT SEQ ID NO: 166 MacMul-Xa-Fc-His TPP-1792 hCeacam6-Dom2-Chain 1 PRT SEQ ID NO: 167 MacMul-Xa-Fc-His TPP-1793 hCeacam6-Dom3-Chain 1 PRT SEQ ID NO: 168 MacMul-Xa-Fc-His TPP-1794 hCeacam6-Dom1-Chain 1 PRT SEQ ID NO: 169 8xHis (E. coli) TPP-2443 cyno CEACAM-6- Chain1 PRT SEQ ID NO: 170 Xa-Fc-His TPP-2452 cynomolgus Chain 1 PRT SEQ IDNO: 171 Ceacam6-Dom1- Xa-Fc-His TPP-2755 human CEACAM3 Chain 1 PRT SEQID NO: 172 TPP-4185 CEACAM1 Chain 1 PRT SEQ ID NO: 173 TPP-4186 CEACAM19Chain 1 PRT SEQ ID NO: 174 TPP-4187 CEACAM3 Chain 1 PRT SEQ ID NO: 175TPP-4188 CEACAM5 Chain 1 PRT SEQ ID NO: 176 TPP-4189 CEACAM6_macfa Chain1 PRT SEQ ID NO: 177 TPP-4190 CEACAM8 Chain 1 PRT SEQ ID NO: 178TPP-4639 CEACAM6 Chain 1 PRT SEQ ID NO: 179 TPP-2453 Macaca fascicularisChain 1 PRT SEQ-ID NO: 180 CEACAM6-Domain 1-His TPP-5468 TPP-2971HU1-Heavy Chain PRT SEQ-ID NO: 181 hIgG1Kappa TPP-5468 TPP-2971HU1- LightChain PRT SEQ-ID NO: 182 hIgG1Kappa APP-1574 Papain-cleaved Fab HeavyChain PRT SEQ-ID NO: 183 fragment of TPP- 5468 APP-1574 Papain-cleavedFab Light Chain PRT SEQ-ID NO: 184 fragment of TPP- 5468 TPP-8697 HumanCEACAM6- Chain 1 PRT SEQ-ID NO: 185 Domain 1-His- I30L TPP-8698 HumanCEACAM6- Chain 1 PRT SEQ-ID NO: 186 Domain 1-His- I30F

1. An isolated antibody or antigen-binding fragment thereof specificallybinding to human CEACAM6 and Macaca fascicularis CEACAM6.
 2. Theantibody or antigen-binding fragment thereof according to claim 1binding to CEACAM6 domain 1 represented by amino acids 35-142 of SEQ-IDNO:179 and amino acids 35-142 of SEQ-ID NO:177.
 3. The antibody orantigen-binding fragment thereof according to claim 1 which does notsignificantly cross-react with human CEACAM1, human CEACAM3, and humanCEACAM5.
 4. The antibody or antigen-binding fragment thereof accordingto claim 1 which interferes with the CEACAM6 and CEACAM1 interaction. 5.The antibody or antigen-binding fragment thereof according to claim 1which is able to change the cytokine profile of tumor antigen specific Tcells towards a more activated phenotype characterized by an IFN-gammasecretion increase, preferably by a ≥1.5 times increase compared tocontrol samples.
 6. The antibody or antigen-binding fragment thereofaccording to claim 1 which binds to an epitope of human CEACAM6, whereinsaid epitope comprises one or more amino acid residues selected from thegroup consisting of Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85,Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 17
 7. The antibody orantigen-binding fragment thereof according claim 6 which binds to anepitope of human CEACAM6, wherein said epitope comprises the amino acidresidues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127,Asp128 and Leu129 of SEQ ID NO:
 179. 8. The antibody or antigen-bindingfragment thereof according to claim 6 which binds to a human CEACAM6protein comprising an Ile63Leu mutation and which does not bind to ahuman CEACAM6 protein comprising an Ile63Phe mutation according to SEQID NO:
 179. 9. The antibody or antigen-binding fragment thereofaccording to claim 1 comprising i. a heavy chain antigen-binding regionthat comprises an H-CDR1 comprising SEQ ID NO:48, an H-CDR2 comprisingSEQ ID NO:49, and an H-CDR3 comprising SEQ ID NO:50 and a light chainantigen-binding region that comprises a L-CDR1 comprising SEQ ID NO:52,a L-CDR2 comprising SEQ ID NO:53, and a L-CDR3 comprising SEQ ID NO:54,or ii. a heavy chain antigen-binding region that comprises an H-CDR1comprising SEQ ID NO:106, an H-CDR2 comprising SEQ ID NO:107, and anH-CDR3 comprising SEQ ID NO:108 and a light chain antigen-binding regionthat comprises a L-CDR1 comprising SEQ ID NO:110, a L-CDR2 comprisingSEQ ID NO:111, and a L-CDR3 comprising SEQ ID NO:112, or iii. a heavychain antigen-binding region that comprises an H-CDR1 comprising SEQ IDNO:4, an H-CDR2 comprising SEQ ID NO:5, and an H-CDR3 comprising SEQ IDNO:6 and a light chain antigen-binding region that comprises a L-CDR1comprising SEQ ID NO:8, a L-CDR2 comprising SEQ ID NO:9, and a L-CDR3comprising SEQ ID NO:10, or iv. a heavy chain antigen-binding regionthat comprises an H-CDR1 comprising SEQ ID NO:34, an H-CDR2 comprisingSEQ ID NO:35, and an H-CDR3 comprising SEQ ID NO:36 and a light chainantigen-binding region that comprises a L-CDR1 comprising SEQ ID NO:38,a L-CDR2 comprising SEQ ID NO:39, and a L-CDR3 comprising SEQ ID NO:40,or v. a heavy chain antigen-binding region that comprises an H-CDR1comprising SEQ ID NO:120, an H-CDR2 comprising SEQ ID NO:121, and anH-CDR3 comprising SEQ ID NO:122 and a light chain antigen-binding regionthat comprises a L-CDR1 comprising SEQ ID NO:124, a L-CDR2 comprisingSEQ ID NO:125, and a L-CDR3 comprising SEQ ID NO:126, or vi. a heavychain antigen-binding region that comprises an H-CDR1 comprising SEQ IDNO:24, an H-CDR2 comprising SEQ ID NO:25, and an H-CDR3 comprising SEQID NO:26 and a light chain antigen-binding region that comprises aL-CDR1 comprising SEQ ID NO:28, a L-CDR2 comprising SEQ ID NO:29, and aL-CDR3 comprising SEQ ID NO:30, or vii. a heavy chain antigen-bindingregion that comprises an H-CDR1 comprising SEQ ID NO:76, an H-CDR2comprising SEQ ID NO:77, and an H-CDR3 comprising SEQ ID NO:78 and alight chain antigen-binding region that comprises a L-CDR1 comprisingSEQ ID NO:80, a L-CDR2 comprising SEQ ID NO:81, and a L-CDR3 comprisingSEQ ID NO:82, or viii. a heavy chain antigen-binding region thatcomprises an H-CDR1 comprising SEQ ID NO:134, an H-CDR2 comprising SEQID NO:135, and an H-CDR3 comprising SEQ ID NO:136 and a light chainantigen-binding region that comprises a L-CDR1 comprising SEQ ID NO:138,a L-CDR2 comprising SEQ ID NO:139, and a L-CDR3 comprising SEQ IDNO:140, or ix. a heavy chain antigen-binding region that comprises anH-CDR1 comprising SEQ ID NO:148, an H-CDR2 comprising SEQ ID NO:149, andan H-CDR3 comprising SEQ ID NO:150 and a light chain antigen-bindingregion that comprises a L-CDR1 comprising SEQ ID NO:152, a L-CDR2comprising SEQ ID NO:153, and a L-CDR3 comprising SEQ ID NO:154, or x. aheavy chain antigen-binding region that comprises an H-CDR1 comprisingSEQ ID NO:14, an H-CDR2 comprising SEQ ID NO:15, and an H-CDR3comprising SEQ ID NO:16 and a light chain antigen-binding region thatcomprises a L-CDR1 comprising SEQ ID NO:18, a L-CDR2 comprising SEQ IDNO:19, and a L-CDR3 comprising SEQ ID NO:20, or xi. a heavy chainantigen-binding region that comprises an H-CDR1 comprising SEQ ID NO:62,an H-CDR2 comprising SEQ ID NO:63, and an H-CDR3 comprising SEQ ID NO:64and a light chain antigen-binding region that comprises a L-CDR1comprising SEQ ID NO:66, a L-CDR2 comprising SEQ ID NO:67, and a L-CDR3comprising SEQ ID NO:68, or xii. a heavy chain antigen-binding regionthat comprises an H-CDR1 comprising SEQ ID NO:92, an H-CDR2 comprisingSEQ ID NO:93, and an H-CDR3 comprising SEQ ID NO:94 and a light chainantigen-binding region that comprises a L-CDR1 comprising SEQ ID NO:96,a L-CDR2 comprising SEQ ID NO:97, and a L-CDR3 comprising SEQ ID NO:98.10. The antibody or antigen-binding fragment thereof according to claim1 comprising i. a variable heavy chain sequence as presented by SEQ IDNO: 47 and a variable light chain sequence as presented by SEQ ID NO:51, or ii. a variable heavy chain sequence as presented by SEQ ID NO:105 and a variable light chain sequence as presented by SEQ ID NO: 109,or iii. a variable heavy chain sequence as presented by SEQ ID NO: 3 anda variable light chain sequence as presented by SEQ ID NO: 7, or iv. avariable heavy chain sequence as presented by SEQ ID NO: 33 and avariable light chain sequence as presented by SEQ ID NO: 37, or v. avariable heavy chain sequence as presented by SEQ ID NO: 119 and avariable light chain sequence as presented by SEQ ID NO: 123, or vi. avariable heavy chain sequence as presented by SEQ ID NO: 23 and avariable light chain sequence as presented by SEQ ID NO: 27, or vii. avariable heavy chain sequence as presented by SEQ ID NO: 75 and avariable light chain sequence as presented by SEQ ID NO: 79, or viii. avariable heavy chain sequence as presented by SEQ ID NO: 133 and avariable light chain sequence as presented by SEQ ID NO: 137, or ix. avariable heavy chain sequence as presented by SEQ ID NO: 147 and avariable light chain sequence as presented by SEQ ID NO: 151, or x. avariable heavy chain sequence as presented by SEQ ID NO: 13 and avariable light chain sequence as presented by SEQ ID NO: 17, or xi. avariable heavy chain sequence as presented by SEQ ID NO: 61 and avariable light chain sequence as presented by SEQ ID NO: 65, or xii. avariable heavy chain sequence as presented by SEQ ID NO: 91 and avariable light chain sequence as presented by SEQ ID NO:
 95. 11. Theantibody according to claim 1, which is an IgG antibody.
 12. Theantibody according to claim 1 comprising: i. a heavy chain regioncorresponding to SEQ ID NO: 57 and a light chain region corresponding toSEQ ID NO: 58, or ii. a heavy chain region corresponding to SEQ ID NO:115 and a light chain region corresponding to SEQ ID NO: 116, or iii. aheavy chain region corresponding to SEQ ID NO: 43 and a light chainregion corresponding to SEQ ID NO: 44, or iv. a heavy chain regioncorresponding to SEQ ID NO: 129 and a light chain region correspondingto SEQ ID NO: 130, or v. a heavy chain region corresponding to SEQ IDNO: 85 and a light chain region corresponding to SEQ ID NO: 86, or vi. aheavy chain region corresponding to SEQ ID NO: 143 and a light chainregion corresponding to SEQ ID NO: 144, or vii. a heavy chain regioncorresponding to SEQ ID NO: 157 and a light chain region correspondingto SEQ ID NO: 158, or viii. a heavy chain region corresponding to SEQ IDNO: 71 and a light chain region corresponding to SEQ ID NO: 72, or ix. aheavy chain region corresponding to SEQ ID NO: 101 and a light chainregion corresponding to SEQ ID NO:
 102. 13. The antigen-binding fragmentaccording to claim 1, which is an scFv, Fab, Fab′ fragment or a F(ab′)2fragment.
 14. The antibody or antigen-binding fragment according toclaim 1, which is a monoclonal antibody or antigen-binding fragment. 15.The antibody or antigen-binding fragment according to claim 1, which ishuman, humanized or chimeric antibody or antigen-binding fragment. 16.An antibody-drug conjugate, comprising an antibody or antigen bindingfragment thereof according to claim
 1. 17. An isolated nucleic acidsequence that encodes the antibody or antigen-binding fragment accordingto claim
 1. 18. A vector comprising a nucleic acid sequence according toclaim
 17. 19. An isolated cell expressing an antibody or antigen-bindingfragment according to claim
 1. 20. An isolated cell according to claim19, wherein said cell is a prokaryotic or a eukaryotic cell.
 21. Amethod of producing an antibody or antigen-binding fragment according toclaim 1 and purification of the antibody or antigen-binding fragment.22. An antibody or antigen-binding fragment according to claim 1 for useas a medicament.
 23. An antibody or antigen-binding fragment accordingto claim 1 for use as a diagnostic agent.
 24. An antibody orantigen-binding fragment according to claim 1 for use as a medicamentfor the treatment of cancer.
 25. A pharmaceutical composition comprisingan antibody or antigen-binding fragment according to claim
 1. 26. Acombination of a pharmaceutical composition according to claim 25 andone or more therapeutically active compounds.
 27. A method for treatinga disorder or condition associated with the undesired presence ofCEACAM6, comprising administering to a subject in need thereof aneffective amount of the pharmaceutical composition according to claim25.
 28. A process for the preparation of anti-CEACAM6 antibodiesspecifically binding to human CEACAM6 and Macaca fascicularis CEACAM6,which process comprises immunization of an animal, preferentially amouse, with a protein comprising cynomolgus CECAM6 domain 1 representedby amino acids 35-142 of SEQ-ID NO:177, determining the amino acidsequence of antibodies specifically binding to human CEACAM6 and tocynomolgus CEACAM6, followed optionally by humanization or generation ofa chimeric antibody, and recombinant expression of said antibodies.